Compositions and methods for treating viral infections

ABSTRACT

Methods and compositions for treatment, diagnosis, and prevention of a virus comprise administering to a patient antibodies which react with regions of viral proteins and result in neutralization of infectivity and inactivation of functionally essential events in the life cycle of the virus. The antibodies recognize viral epitopes which fail to elicit an immune response in man when encountered through infection or naturally through the environment. In a preferred embodiment, the invention provides compositions and methods useful in the treatment and diagnosis of human immunodeficiency virus (HIV) infections.

This application is a divisional application of U.S. Ser. No.09/482,612, filed Jan. 14, 2000 is now U.S. Pat. No. 6,258,599, which isa divisional of U.S. Ser. No. 08/948,782 is now 6,043,347, filed Oct.10, 1997 which claims benefit of 60/028,194 Oct. 10, 1996.

TECHNICAL FIELD

The present invention relates generally to the treatment and preventionof viral infections. In particular, the invention provides compositionsand methods for the production of antibodies and peptides useful in thetreatment and diagnosis of human immunodeficiency virus (HIV)infections.

BACKGROUND OF THE INVENTION

The diagnosis, treatment and prevention of viral infections is a primaryfocus of many medical researchers. Although compositions and methods ofdiagnosing, treating and vaccinating against a number of viralinfections are known, there are still a number of viruses which aredifficult to detect in man and for which no effective methods oftreatment or vaccination against are known. Of these, one of the mostsignificant, of course, is HIV.

The infectious agent responsible for acquired immunodeficiency syndrome(AIDS) and its prodromal phases, AIDS-related complex (ARC) andlymphadenopathy syndrome (LAS), is a lymphotrophic retrovirus termedLAV, HTLV-III, ARV, and recently HIV as recommended by the InternationalCommittee on Taxonomy of Viruses (Ref 299). Nomenclature herein employsthese recommendations to designated viruses associated with AIDS and thestrains thereof. Historic references to strains, which include LAV andARV-2, are now named HIV1 LAI and HIV1_(SF2), respectively.

As the spread of HIV reaches pandemic proportions, the treatment ofinfected individuals and prevention of the transmission to uninfectedindividuals at risk of exposure is of paramount concern. A variety oftherapeutic strategies have targeted different stages in the life cycleof the virus and are outlined in Mitsuya and Broder, 1987, Nature325:773. One reproach involves the use of antibodies which bind to thevirus and inhibit viral replication, either by interfering with viralentry into host cells or by some other mechanism. Once the viralcomponent(s) susceptible to antibody intervention are identified, it hasbeen hoped that antibody reactivity sufficient to neutralize theinfectivity of the virus could be generated and administered toHIV-infected patients in the form of immune globulins or purifiedantibodies and that this passive immunization procedure would alter orreverse Progression of HIV infection. In addition, it has been hopedthat the vaccination of non-infected individuals with selected epitopesmodified to enhance MHC interactiors would provide protection fromsubsequent infection following exposure to HIV.

The envelope glycoproteins of most retroviruses are thought to reactwith receptor molecules on the surface of susceptible cells, therebydetermining the virus' infectivity for certain hosts. Antibodies thatbind to these envelope glycoproteins may block the interaction of thevirus with the cell receptors, neutralizing the infectivity of thevirus. See generally, The Molecular Biology of Tumor Viruses, 534 (J.Tooze, ed., 1973); and RNA Tumor Viruses, 226, 236 (R. Weiss et al.,eds., 1982); Gonzalez-Scarano et al., 1982, Virology 120:42 (La CrosseVirus); Matsuno and Inouye, 1983, Infect. Immun. 39:155 (Neonatal CalfDiarrhea Virus); and Mathews et al., 1982, J. Immunol., 129:2763(Encephalomyelitis Virus). To date, therapeutic strategies directed ateliciting protective immune responses in man by vaccination with HIVproteins/peptides have failed. In addition, neither high titerneutralizing antibodies recovered from HIV-infected patients normonoclonal antibodies produced in mice have succeeded in altering theprogression of HIV infection to AIDS and death. There is a need in theart to identify alternate immunological targets on HIV which will elicitimmune responses that will modify the course of HIV infection.

The general structure of HIV is that of a ribonucleo-protein coresurrounded by a lipid-containing envelope which the virus acquiresduring the course of budding from the membrane of the infected hostcell. Embedded within the envelope and projecting outward are the viralencoded glycoproteins. The envelope glycoproteins of HIV are initiallysynthesized in the infected cell as a precursor molecule of150,000-160,000 Daltons (gp 160), which is then processed in the cellinto an N-terminal fragment of 110,000-120,000 Daltons (gp 120) togenerate the external glycoprotein, and a C-terminal fragment of41,000-46,000 Daltons (gp 41), which is the transmembrane envelopeglycoprotein.

For the reasons discussed above, the gp 120 glycoprotein of HIV has beenthe object of much investigation as a potential target for interruptingthe virus' life cycle. Sera from HIV-infected individuals have beenshown to neutralize HIV in vitro, and antibodies that bind to purifiedgp 120 are present in these sera, (Robert-Guroff et al., 1985, Nature316:72; Weiss et al., 1985, Nature 316:69; and Mathews et al., 1986,Proc. Natl. Acad. Sci. U.S.A., 83:9709). Purified and recombinant gp 120stimulated the production of neutralizing serum antibodies when used toimmunize animals (Robey et al., 1986, Proc. Natl. Acad. Sci. U.S.A.,83:7023; Lasky et al., 1986, Science, 233:209) and a human (Zagury etal., 1986, Nature 326:249). Binding of the gp 120 molecule to the CD4receptor also has been shown and monoclonal antibodies which recognizecertain epitopes of the CD4 receptor have been shown to block HIVbinding, syncytia formation, and infectivity. McDougal et al., (1986,Science 231:382) and Putney et al. (1986, Science 234:1392) elicitedneutralizing serum antibodies in animals after immunizing with arecombinant fusion protein containing the carboxyl-terminal half of thegp 120 molecule and further demonstrated that glycosylation of theenvelope protein is unnecessary for a neutralizing antibody response.

Shortly after HIV infection the immune system of man responds to thevirus with both antibody production and cell mediated immune responses.A review of the immune responses to retroviruses has been published(Norley, S., and Kurth R., 1994: The Retroviridae, Vol E, J. A. Levy,ed., pp. 363-464, Plenum Press). Human antibodies specific for a numberof HIV proteins including gp 160, gp 120, p66, p55, gp 41, p32, p24, andp17 have been reported (Carlson, 1988, J.Am. Med. Assoc. 206:674). Theinitial antibody response in man to HIV is directed to p17 and p24,followed by gp 120/160, then by gp 41, p66/55 and finally p32 (Lange, Jet al 1986, Br. Med. J. 292:228). As HIV infection progresses into AIDSantibody levels to p17 and p24 markedly fall to undetectable limits andare replaced by p17 and p24 antigenemia. Antibody titers to p32 and p55also decline but to a lesser degree (McDougal et al 1987 J. Clin.Invest. 80:316). However, substantial amounts of antibodies to gp160/120 persist throughout the entire course of HIV infection. Duringthe early phases of HIV infection an elevation in total immunoglobulinsis observed and this increased quantity of antibody is specific for HIVand predominantly directed to gp 120, (Amadori et al., 1988 Clin.Immunol. Immunopathol. 46:342; Amadori et al., 1989, J. Immunol143:2146). Possible mechanisms for this HIV specific hyper gammaglobulinemia have been reviewed by Barker E. et al 1995: TheRetroviridae Vol 4, J. A. Levy, ed. pp 1-96 Plenum Press. Functionalproperties and epitopes targeted by these antibodies produced during HIVinfection have been described and include epitopes which are susceptibleto antibody mediated neutralization. These primary target epitopes areprimarily located on the envelope protein gp160 (gp120/gp41) and the gagprotein p17; for review see Levy, 1994 Am.Soc. Micro; Nixon et al, 1992Immunol 76:515. Neutralizing antibodies to HIV envelope protein havebeen identified and bind to conserved and divergent domains on gp 120.These include regions localized to the CD4 binding regions Linsley et al1988 and Thali et al, 1992); the second and third variable loop domains(Fung et al, 1992 and Haigwood et al 1990); and carbohydrate moieties(Benjouad et al, 1992 and Feizi and Larkin, 1990). Other neutralizationsites have been identified on the external portion of gp 41 and abinding site on p17 (Changh et al, 1986). Early studies suggested thatthe presence of neutralizing antibodies lead to a more favorableclinical outcome, (Robert-Guroff et al, 1985). However, these studiesemployed selected sera with high neutralizing capacity againstlaboratory strains of HIV and not against autologous HIV isolates (Homsyet al, 1990; Tremblay and Wainberg, 1990). Subsequent investigationdemonstrated that autologous antibody had little or no neutralizingactivity against autologous HIV isolates (Homsy et al, 1990). The lackof susceptibility to antibody mediated neutralization in the presence ofa neutralizing antibody is thought to result from the development ofescape mutants that appear after seroconversion (Arendrup et al, 1992)and throughout the infection as new antibody specificities are produced.The clinical relevance of neutralizing antibodies produced as aconsequence of HIV infection is unclear. However, it is clear that inspite of a vigorous immune response to HIV in individuals infected withHIV, progress to AIDS and, ultimately, death as a consequence of immunedysfunction predominates. Accordingly, new methods of treatment aresought.

OBJECTS OF THE INVENTION

It is an object of this invention to identify neutralizing regions ofviral proteins which fail to elicit immune responses in man but doelicit immune responses in non-human mammals and to produce antibodiesreactive with these regions. It is a further object of this invention touse these identified neutralizing regions of proteins and the antibodiesreactive with them in the diagnosis, treatment and prevention of diseasecaused by the virus. Further objects of this invention will be apparentfrom the description of the invention detailed below.

SUMMARY OF THE INVENTION

In accordance with the present invention there are provided methods andcompositions for the treatment, diagnosis and prevention of viralinfection by the use of antibodies which react with regions of viralproteins to neutralize and inactivate functionally essential events inthe life cycle of the virus. The antibodies recognize viral epitopeswhich fail to elicit an immune response in humans when encounteredthrough infection or through environmental exposure but do elicit animmune response in non-human mammals.

Selected epitopes that react with non-human anti-viral antibodies butnot with human anti-viral antibodies are identified. These epitopesescape surveillance by the human immune system through molecular mimicryto human proteins and in some instances are composed of amino acidssusceptible to enzymatic cleavage in antigen processing cells. Desiredepitopes are enzymatically cleaved by human enzymes and therefore arenot processed for immune presentation.

Peptides representing these epitopes can be synthesized, optionallymodified, and conjugated to a macrocarrier adjuvant to elicit antibodyresponses in non-humans. The preferred adjuvant is a microparticlecomprising multiple repeats of muramyl dipeptide extracted fromPropionibacterium acini.

Antibodies and peptides of this invention can be used in immunoassayconfigurations to identify species specific epitopes and to quantitateviral antigens in human tissues and fluids. In a preferred embodiment,the invention provides antibody and peptide compositions and methodsuseful in the treatment and diagnosis of individuals infected with thevirus.

In a preferred embodiment, the virus of interest is HIV.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compositions and methods fordiagnosing and neutralizing viral infections. The invention will bedescribed in detail with a focus on a preferred embodiment, in which thevirus of interest is HIV. It is to be understood, however, that theprinciples of the invention can be used to identify neutralizing regionsof proteins of other viruses and to produce antibodies reactive withthose proteins that can be used to diagnose, treat and preventinfections caused by these other viruses as well.

Focusing now on HIV, this invention provides novel compositions andmethods for neutralizing HIV infection and preventing or substantiallyinhibiting HIV infectivity, cell to cell transmission, and virusproduction in the infected host. More specifically, HIV proteinsequences containing epitopes which fail to elicit an immune response inman when encountered through infection or naturally through theenvironment are utilized as described in detail below to produceantibodies in non-human mammals which can be administered to neutralizeHIV infectivity, facilitate killing of infected CD4 lymphocytes, andinactivate essential steps in the life cycle of HIV. The term“neutralizing region” indicates those portions of HIV, particularly HIVproteins, containing amino acid segments defining one or more epitopesreactive with antibodies which, either individually or in combinationwith other antibodies of the present invention, are capable ofneutralizing HIV infections. Suitable assays for evaluatingneutralization are well known and can include assays which measurereduction of HIV infections in T-cell lines, reduction of plaque formingunits of VSV (HIV) pseudo types bearing the envelope glycoproteins ofHIV, syncytial inhibition tests, and virion-receptor binding tests. Theterm “inactivating region” indicates those segments of HIV proteinswhich contain one or more epitopes which when reacted with antibodies ofthis invention, either individually or in combination, inactivatefunctionally important events in the life cycle of HIV. Suitable assaysto evaluate antibody-mediated destruction of HIV infected lymphocytesare well known and can include antibody dependent cell mediatedcytotoxicity, complement mediated lysis, and natural killer (NK) assays.Suitable assays for measuring antibody-mediated inactivation ofessential steps of the life cycle of HIV include assays which determineinactivation of reverse transcriptase, or measure polymerase andprotease activity, or which evaluate antibody-mediated complementdependent changes in nuclear capsid permeability exposing viral RNA toribonuclease degradation. As desired, the neutralizing activity can becompared to antibody reactivity in immunochemical assays, such asimmunofluorescence, immunoblot, enzyme linked immunoassay, andradioimmunoassay.

The present invention is based on the discovery that epitopes which arefunctionally important in the life cycle of HIV, but not immunogenic inman, can be identified and characterized employing antibodies producedin selected mammalian species other than man. In addition, it has beendiscovered that the immunological non-responsiveness in man to theseregions is a function of molecular mimicry and lack of MHC associatedevents, including antigen presentation through required MHC HLA class 1and HLA class 2 associated events. With molecular mimicry the epitope isseen as self and is not responded to under normal circumstances. Withlack of MHC associated events in antigen presentation several steps areinvolved and failure of any one step can result in the absence of animmunological response to the antigen.

Peptide regions containing multiple overlapping epitopes and antibodiesto these epitopes have been produced and are shown to neutralize andinactivate essential steps in the life cycle of HIV in vitro. Inaddition, HIV-infected patients with AIDS have been treated withantibodies to these peptide regions, resulting in rapid reduction inblood born infectivity measured by total culturable infectious dose(TCID). Treatment of chronic AIDS patients with these antibodies hasresulted in marked clinical improvement, including weight gain,resolution of opportunistic infections, decreased incidence and severityof infections and doctor visits and resolution of HIV-associatedneuropathy. The patients further have shown immunological recovery, asdefined by a reduction of HIV-RNA, increased CD4 number, increased CD8number and restoration of the cytokine system associated with improvedCD4 and CD8 numbers and function.

Identification of Epitopes and Production of Antibodies

The majority of immune reactions target immunodominant epitopes. HIVepitopes are most frequently identified and mapped by variousimmunological methods which employ antisera to HIV, cytotoxic Tlymphocyte reactivity to HIV epitope targets and helper lymphocyteantigen presentation of HIV epitopes. Synthetic peptides of knownsequence which mimic HIV sequence can be employed competitively ornoncompetitively using well known assays to confirm these observations.It is to be understood that stimulation of the immune system can leadeither to enhancement or suppression of the immune response. Factorswhich govern this include:

A. The sub population of lymphocytes stimulated by the immunogen(Suppressor versus Helper).

B. The micro-environment, including cell population residing therein,which contact the immunogen first.

C. The type of cytokines present in the micro-environment at the time ofeffector cell contact with the immunogen.

D. The type of cytokines elicited following effecter cell contact withthe immunogen.

E. Structural and biochemical composition of the immunogen.

F. The amino acid sequence of the immunogen and its susceptibility toprotease degradation by proteases in the micro environment.

From preliminary experiments, the following properties were determinedto be fundamentally important in determining the potential value ofcertain proteins and peptides for use in the production of antibodieswhich are intended for passive immunotherapy application in thetreatment or palliation of disease processes in man and, in particular,infection with HIV at all stages including AIDS:

A. The immunogen must lack epitope determinants that are expressed onhuman cells and tissues when employed to produce antibody for use inpassive immunotherapy with the following exceptions:

1. Antigen distribution is restricted to sequestered and/or unavailablelocations to the antibody.

2. The antigen is expressed during developmental phases which permit theuse of antibody at specific times during the developmental cycle, whenantigen is not available.

3. Epitope location within the host is not adjacent to a vitalstructure.

4. Antigen distribution on host cell is at a density lower than requiredto produce injury but favorable on the desired target resulting inselective targeting.

5. The target to normal ratio must be sufficiently different and favorantibody delivery to the desired target.

B. The number of peptide repeats delivered to an antigen-presenting celldirectly influences the magnitude of the immunological response.

C. The epitope must not be present in body fluids at concentrationswhich would neutralize antibody and prevent targeting.

To date vaccine development has focused on designing better technologyto amplify responses to targets to which the immune system of manresponds, and passive immunotherapy has resulted in inconclusiveresults. Disclosed herein are alternate targets on HIV which do notelicit immune events in man as well as a new configuration to deliverantigen which results in immune reactions to HIV not previouslyattainable. The methods disclosed herein focus on the treatment of HIVand AIDS, but it is to be understood that the formulations of thisinvention have broad application. The antibody response in goatsdemonstrates utility of invention by way of antibody production to keytargets on HIV, and by way of treatment which results in clinicalimprovement of AIDS. This technology has broad application in vaccinedevelopment.

Successful immune induction to antigen challenge requires thepresentation of multiple epitope repeats by an antigen-presenting cell(APC) through MHC events. Epitopes which are most immunogenic are in anamphipathic configuration with a hydrophobic amino acid on one terminus,a hydrophillic amino acid on the other terminus, contain amino acidsconsistent with the formation of amphipathic helices; i.e., they lackhelix breaking amino acids, such as proline, and lack carbohydrate.Sequences which lack amino acids that are susceptible to proteasedegradation by proteases in the micro-environment are especiallydesired.

To identify immunological targets on HIV with functional importance,immunogenic regions on HIV-related proteins in animal species other thanman were determined. Goats were immunized with purified HIV lysate withand without carbohydrate groups removed. Removing carbohydrate residuesfrom HIV proteins has little effect on the immunological response to theproteins yet can expose hidden epitopes. HIV lysates obtainedcommercially were further purified to remove proteins of tissue cultureorigins including human HLA class 1 antigens, HLA class 2 antigens, andbeta-2-microglobulin. Following immunization, antisera from the goatswere tested employing competitive immunoassay methodology to identifyHIV peptides not recognized by antibodies pooled from HIV-infectedpatients. Pools of human HIV antisera were prepared from selectedpatient sera with high neutralizing and Western Blot activity andemployed as competitive antibodies using standard competitiveimmunoassay methods. A broad spectrum of goat antibodies were identifiedwhich reacted with HIV determinants immunologically distinct from thoserecognized by the human anti-HIV antisera pools.

Those skilled in the art recognize that other animal species could beused to produce antibodies to these epitopes and that such antibodiescould function in ADCC and complement mediated reactions. Other suitableanimal species for the production of antibodies include, but are notlimited to, sheep, rabbits, horses, cows and mice.

The epitope reactivity of the anti-HIV antibodies was characterizedusing twelve-mer peptides spanning the linear amino acid sequences ofHIV1_(SF2). Peptides of this size react well with antibodies, can besynthesized easily and can be prepared in highly purified form. Peptideswere synthesized by and purchased from Purification Systems, Inc. Thesynthetic peptides were combined with peroxidase labeled goat anti-HIVantibodies and combined with each of two sets of microtiter wells coatedwith HIV. One set was blocked with human IgG anti-HIV; the other set wasnot. The percent of peptide inhibition of goat anti-HIV binding to HIVprotein sites blocked with human anti-HIV was determined.

When inhibition of binding was observed with a specific syntheticpeptide, additional peptides were synthesized with amino acid sequencesoverlapping that of the original inhibiting peptide to further definethe epitope sequences.

The location of the epitopes on the HIV proteins recognized by goatanti-HIV IgG but not human anti-HIV IgG were further evaluated andconfirmed using HIV peptide-HRP conjugates as the identificationmarkers. In this assay, HIV proteins were absorbed to supports such asmicrotiter plate wells or precision polystyrene beads. Twelve-merpeptides spanning the linear amino acid sequence of HIV1_(SF2) werecovalently attached to horseradish peroxidase. Human and goat anti-HIVreactivity were measured independently with anti-HIV reactivity fromhuman and goat bridging the native epitopes adsorbed to the support andto the peptide epitope covalently attached to peroxidase. With thisprocedure, detailed in Example 8, only exact epitopes contained withinthe synthetic peptide were recognized.

Once peptides having significant mimicry with human proteins have beenidentified, those sequences which have functional importance in the lifecycle of HIV are determined. This is done, as described below andillustrated in Example 8, by generating antibodies to candidate peptidesand then testing those antibodies for their effects on HIV infectivityand viral neutralization.

As noted above, a number of specific epitope regions have beenidentified and nine are described in detail below with reference to theHIV1_(SF2) sequence unless otherwise indicated. Amino acid residuedesignations set forth below and Throughout this application forHIV1_(SF2) are from the Los Alamos Data Bank (AIDS Virus Sequence DataBase, Los Alamos National Laboratories, Theoretical Division, LosAlamos, N. Mex. 87545). Amino acid residue designations set forth belowand throughout this application for HIV2_(NZ) are from the Ex Pasy WorldWide Web Molecular Biology Server of the Geneva University Hospital andthe University of Geneva, and the BioAccelerator available throughCompugen Ltd. at the Weizman Institute, Israel, and Akira Ohyama,BioScience Systems Department, Mitsuey Knowledge Industry Co., Ltd.,Tokyo, Japan. Those skilled in the art will appreciate that additionalanalogous regions (“homologs”) from other HIV isolates can be identifiedbased upon their location within related proteins from various isolates.In practice, such homologs can be identified by reference to HIV1_(SF2)sequence data as follows:

(a) the amino acid sequences of HIV isolates and HIV1_(SF2) can bealigned to obtain maximum homology between the two sequences, generallyat least about 75% identify between the sequences;

(b) once an amino acid sequence is aligned to the corresponding locationwithin HIV1_(SF2) proteins will demonstrate immunological mimicry,similarity, or identity with HIV1_(SF2) as defined by retention ofantibody reactivity to the mimicked or homologous sequence. Peptidesfrom other HIV isolates and their amino acid sequences so identifiedtypically will immunologically mimic corresponding regions onHIV1_(SF2).

This method of identifying key epitopes can be applied to HIV strainsthat are yet to be discovered. For example, as new strains of HIV areidentified, their envelope and core amino acid sequences can be alignedwith that of HIV1_(SF2) to obtain maximum sequence homology with thatstrain. The methods by which the sequences are aligned are known tothose skilled in the art. In aligning the sequences it is desired tomaintain as much homology between cysteine residues as possible. Theamino acid sequence(s) of the new HIV strain or species whichcorresponds to the location of the peptides specifically disclosedherein can be synthesized and used in accordance with the invention.

It is not necessary to the present invention that the epitopes containedwithin such sequences be cross-reactive reactive with antibodies to allstrains or species of HIV. Peptides encompassing immunological epitopeswhich distinguish one species or serogroup over another will findutility in identifying particular species or serogroups and may assistin identifying individuals infected with one or more species orserogroups of HIV. They also can be useful in combination with otherpeptides, from either a homologous region or another neutralizingregion, in therapeutic regimens.

The amino acid sequences of this invention typically comprise from about5 to about 50 amino acids and comprise an epitope region or multipleepitope regions located on HIV proteins that fail to elicit a protectiveimmune response in man when encountered through infection orenvironmental contact but do elicit a response in a non-human mammal.Preferably, the sequences comprise between about 5 and 35 amino acids.Synthetic peptides or treated lysates of natural HIV proteins containingthe desired amino acid sequences are used to immunize animals whichrespond immunologically to them and produce antibodies which havetherapeutic value in treating HIV infections.

The amino acid sequences or peptides of interest fail to elicit animmune response in man through mimicry of epitopes on human and otherproteins. Of particular interest are peptide epitopes shared between HIVproteins and human alpha fetoprotein, aspartyl protease, deoxyuridine5′-triphosphate nucleotidohydrolase, eosinophil cationic protein,eosinophil-derived neurotoxin and ribonuclease 4 precursor and peptideepitope regions mimicked by neurotoxins from Bungaris Naja, Dendoaspis,Psudechis, or Androctonus Centruroides.

In the discussion which follows, reference is made to a number of humanproteins and neurotoxins using standard identifying abbreviations forthe proteins. Set forth below is a table which sets forth theseabbreviations and the full names of the proteins to which theycorrespond:

Human Proteins With Sequence Similarity to HIV Proteins Swiss Prot IDXXXX-Human Protein ACE angiotensin-converting enzyme precursor ACHEacetyl choline receptor protein 3BH1 3-beta hydroxy-5-ene steroiddehydrogenase type I 3BH2 3-beta hydroxy-5-ene steroid dehydrogenasetype II 41BL 4-1BB ligand BLSA beta-lymphocyte antigen precursor CATDcathepsin D precursor CD69 early activation antigen CD69 CD81 CD81antigen CO02 tumor-associated antigen CO-029 CP11 cytochrome P450 IA1CYRP cytokine receptor common beta-chain precursor CYPC peptidyl-prolylcis-trans isomerase C DIAC di-N-acetylchitobiase precursor DUTdeoxyuridine 5-triphosphate nucleotidohydrolase ECP eosinophil cationicprotein precursor EV2B ectotropic viral integration site 2B protein FETAalpha fetoprotein FOL1 folate receptor alpha precursor GSHR glutathionereductase IL9 interleukin 9 precursor IN19 interferon-inducible protein9-27 INIU interferon-inducible protein I-8U INR2 interferon alpha/betareceptor beta-chain precursor KLTK leukocyte tyrosine kinase receptorprecursor KPCL eta type protein kinase C LBP lipopolysaccharide bindingprotein precursor LCAT lecithin-cholesterol acyltransferase LECHasialoglycoprotein receptor 1 LFA3 lymphocyte function-associatedantigen-3 precursor LMA1 lamanin alpha-1 chain precursor LONNmitochondrial LON protease homolog precursor LONM mitochondrial LONprotease homolog precursor LYOX protein-lysine 6-oxidase precursor MAG1melanoma associated antigen 1 MAG2 melanoma associated antigen 2 MAG3melanoma associated antigen 3 MC5R melanocortin-5 receptor MYSEembryonic myosin heavy chain NOL1 proliferating-cell nucleolar antigenP120 NRM1 natural resistance-associated macrophage protein 1 NT3neurotrophin-3 precursor NTCR sodium and chloride-dependent creatintransporter 1 NXS1-NAJAT cobrotoxin PA2M membrane associtedphospholipase A2 precursor PIP5 phospholipase C-gamma-2 PGDSalpha-platelet derived growth factor precursor PLK proteoglycan linkprotein precursor POL1 retrovirus-related pol polyprotein PSS1phosphatidylserine synthease I RENI renin precursor RNKD nonsecretoryribonuclease precursor S5A2 3-oxo-5-alpha-steroid-4-dehydrogenase 2 SDC1syndecan-1 precursor SDC4 syndecan-4 precursor SEMI1 semenogelin 1protein precursor SON SON protein SPCB erthrocyte spectrin beta-chainSRE1 sterol regulatory element binding protein 1 SYV valyl-tRNAsynthetase TCO2 transcobalin II precursor TGL3 protein-glutamineglutamyltransferase E3 precursor TFPI tissue factor pathway inhibitorprecursor TRFL lactotransferrin precursor TYK2 non-receptortyrosine-protein kinase VPRT retrovirus related protease WNT2 WNT-2protein precusor ZN45 zinc finger protein 45

The amino acid sequences of nine of the highly conserved epitope regionsdiscussed above are provided below. Three of these regions are on theenvelope glycoproteins gp120 (two targets) and gp41 (one target), one ison the reverse transcriptase heterodimer p66/55, and one is on proteasep10. Additional targets are on the Gag precursor (p55/Gag) with sites onp17 (two targets), p24 and p7.

One epitope region on HIV1_(SF2) gp120 extends from amino acid residue 4through 27 and a second extends from amino acid residue 54 through 76 ofHIV1. Antibodies to epitope regions located on gp120 functionsynergistically to effect the release of gp120 from gp41. The release ofgp120 from gp41 is antibody dose dependent and can be demonstrated byneutralization assays, such as TCID, which measure HIV infectivity.

An epitope region of a neutralizing or inactivating region of gp120 ofHIV2_(NZ) also has been determined. The sequence of HIV2_(NZ) envelopeglycoprotein gp120 has been mapped, and from about amino acid residue 7through 43 is a region mimicking a sequence of HIV1_(SF2) gp120 andcertain human proteins. Antibody targeting the region results indissociation of HIV2 gp120 from gp41, which correlates with a reductionin infectivity.

A third HIV envelope glycoprotein target for HIV1_(SF2) was located atamino acid residues 502-541 of gp41 transmembrane glycoprotein. Antibodytargeting of this region in the presence of complement results in anantibody dependent complement mediated lysis of the HIV envelopeglycoprotein and marked reduction in HIV infectivity.

In addition to the envelope glycoprotein epitope regions, another HIV1epitope region of interest includes amino acid residues 254 through 295of the reverse transcriptase heterodimer p66/55. Antibody targeting ofthis region results in an antibody dose dependent reduction in reversetranscriptase activity. Also of interest is the epitope regionencompassing amino acid residues 69-94 of protease p10 . Antibodytargeting of this region results in an antibody dose dependent reductionin protease activity.

The targets on reverse transcriptase and protease are in conservedregions adjacent to the enzyme active site, which is well-known for itsmutation and subsequent resistance to competitive inhibitors. Theantibody-mediated inactivation results from a steric or conformationalchange in the enzyme with secondary loss of activity. This method ofinactivation functions independently and is not influenced by mutationin the enzyme active site and is irreversible.

Also of interest are three epitope regions within the Gag gene.Specifically, amino acid residues 166 through 151 of Gag gene proteinp24, one target at amino acid residues 2 through 23 and a second targetat amino acid residues 89 through 122 of Gag gene protein p17 and aminoacid residues 390 through 410 and 438 through 443 of Gag gene protein p7are useful in this invention. Antibodies targeting these regions resultin disruption of the nuclear capsid following lysis of the HIV envelopeby the antibodies described above. This targeting culminates withexposure of HIV RNA to plasma RNAse degradation. Additionally, thetargets on p17 is exposed on the surface of infected lymphocytesfollowing budding. This provides an additional target for ADCC lysis ofinfected lymphocytes.

One of the specific peptides set forth above, comprising at least oneepitope not recognized by antibodies from HIV-infected patients butrecognized by goat anti-HIV antibodies, is the peptide comprising aminoacid residues 4 through 27 of HIV1_(SF2) envelope gp120 protein andlinear epitope-containing subsequences thereof, which has the followingsequence:

KGTRRNYQHLWRWGTLLLGMLMIC [SEQ ID NO. 1]

This peptide mimics human proteins FOL1, NTCR, PIP5, PSS1, KLTK, MC5R,ECP, INIU, INI9, VPRT, CD69, MYSE, RNKD, ACHE, TCO2, LCAT, MAG1, MAG2,MAG3 and LYOX.

A second epitope region from the HIV1_(SF2) gp120 envelope glycoproteinextends from amino acid residue 54 through 76, which has the sequence:

ASDARAYDTEVHNVWATHACVPT [SEQ ID NO. 2]

This peptide mimics proteins CYRB and SYV.

A third epitope region of interest in the envelope of HIV1_(SF2)εextends from amino acid residue numbers 502 through 541 of glycoproteingp41 . This peptide has the following amino acid sequence:

HIV1_Env502

RVVQREKRAVGIVGAM

FLGFLGAAGSTMGAVS

LTLTVQAR 502-541 [SEQ ID NO. 3]

This peptide mimics human proteins CYPC, TYK2, ACHE, NTCF, NTCR, CD81,41BL, NIDO, GSHR, CO02 and TCO2.

In another specific embodiment, an epitope region of interest is that ofamino acid residues 2 through 23 of the HIV1_(SF2) Gag protein p17. Thispeptide has the sequence:

GARASVLSGGELDRWEKIRLRP [SEQ ID NO. 4]

This peptide mimics human proteins TFPI, PA2M, BLSA, ECP, and FETA andcertain neurotoxins, such as NXS1 and NAJAT. The peptide has ahydrophobic sequence which binds to and targets host cell membrane andfunction mimics cellular translation protein Src.

A second target on HIV1_(SF2) p17 extends from amino acid residue 89through 122. This peptide has the sequence:

LYCVHQRIDVKDTKEALEKIEEEQNKSK. [SEQ ID NO. 5]

This peptide mimics FETA and TRIC.

Another peptide of interest is that of amino acid residues 166 through181 of the Gag gene protein p24 and epitope containing subsequencestherein. This peptide has the sequence:

PEVIPMFSALSEGATP [SEQ ID NO. 6]

This peptide mimics human proteins FETA and TRFL.

A third Gag gene protein epitope region of interest is the peptidehaving amino acid residues 390 through 410 and 438-443 of Gag geneprotein p7 and epitope containing subsequences thereof. This peptide hasthe sequence:

KTVKCFNCGKEGHIAKNCRAP [SEQ ID NO. 7]+KIWSSQ [SEQ ID NO. 8]

This peptide mimics human FETA and RNA binding proteins. This peptidecontains a zinc binding domain which interacts with, and binds to, viralRNA. Antibodies to this region enhance the removal of premature HIVdevoid of envelope following the lysis of infected CD4+lymphocytes.

Also of interest as an epitope region is the peptide of amino acidresidues 69 through 94 of the protease p10 and epitope-containingsubsequences thereof. This peptide has the sequence:

RIGGQLKEALLDTGADDTVLEEMNLP [SEQ ID NO. 9]

This peptide sequence mimics human proteins RENI, BLSA, VPRT and CATD.Antibodies to this sequence inhibit the protease activity of HIV.

A further specific sequence useful in this invention is a sequenceencompassing amino acid residues 254 through 295 of HIV1 reversetranscriptase heterodimer p66/55. This peptide has the sequence:

GLKKKKSVTVLDVGDAYFSVPLDKD

FRKYTAFTIPSINNETP [SEQ ID NO. 10]

This peptide sequence mimics human proteins POL1 and ECP.

As noted above, other strains of HIV also can be used to obtain peptidesand antibodies in accordance with the present invention. Useful peptidesfrom other strains can be determined by comparing and aligning thesequence of another strain to the sequence of HIV1_(SF2) or HIV2_(NZ)and finding that part of the sequence homologous to the epitopes ofinterest identified for HIV1_(SF2) or HIV2_(NZ).

A sequence of interest in HIV2_(NZ) identified by the method of thisinvention is in the env gp120 open reading frame and extends from aminoacid residue numbers 7 through 43. This peptide has the followingsequence:

QLLIAIVLASAYLIHCKQF

VTVFYGIPAWRNASIPLF [SEQ ID NO. 11]

This peptide mimics human proteins IL9, SRE1, NRM1, LBP, NOL1, S5A2,LMA1, LECH, LFA3, KPLC, FETA, 3BH2, 3BH1, INR2 and EV2B.

For example, once the desired amino acid sequences have been identified,antibodies which recognize these sequences are obtained. Such antibodiescan be obtained using proteins containing the peptides isolated from HIVlysates, synthetic peptides, bacterial fusion proteins andproteins/peptides from phylogenetically unrelated sources which containthe desired epitopes.

If viral lysates are to be used, a protein lysate of a single HIV straincan be used, or a mixture of lysates of two or more different strainscan be used. If a mixture of lysates is used, the mixture Cain compriselysates of different HIV1 strains or a combination of at least one HIV1strain and at least one HIV2 strain. A preferred mixture is acombination of lysates from HIV1_(BAL), HIV1_(MN) and HIV2_(NZ).

Viral lysates initially are treated to remove lipids and otherimpurities from the HIV proteins. The HIV protein mixture then istreated to remove contaminants of cell culture origin, including humanleukocyte antigen (HLA), class I and class II antigens. Methods forremoving these antigens are known in the art and include employingmonoclonal anti-HLA class I and anti-HLA class II antibodies andimmunoaffinity procedures; one method is set forth in detail in Example3 below.

In addition, it has been found that carbohydates of the HIV proteinsmust be removed; phylogenically preserved carbohydrates determinantsotherwise would stimulate immune responses when the HIV proteins areadministered to an animal, resulting in the production of antibodieswhich would be cytotoxic against human tissues. The proteins are treatedwith enzymes known to those skilled in the art to remove carbohydrates,including PGNase, neuraminidase and glycosidase. One such method isdescribed in detail in Example 3.

The mixture of treated HIV proteins then can be used to immunize ananimal to produce antibodies to the peptides of interest. Desirably, themixture contains approximately equal amounts of the proteins comprisingthe peptides or epitope regions of interest. That is, desirably they areprovided in proportions of approximately 1:1 and the difference in molarratios between any two peptides is no greater than about 10:1,preferably 3:1.

Alternatively, synthetic peptides can be used as the immunogen. Ifsynthetic peptides are used the amino acid sequence of any desiredpeptide can be modified by, for example, using a substituted ortruncated form of the amino acid sequence.

Amino acid substitutions can be made to avoid predicted enzymaticcleavage that can occur during antigen processing at a particular aminoacid moiety, to force amphipathic conformation to meet requiredMHC-associated antigen presentation and to provide sufficient length forHLA presentation should cleavage occur at or near the epitope boundary.Truncated sequences are selected such that the peptide will retainconformity to epitope length requirements as predicted by MHC class 1and class 2 antigen presentation motifs. More extensive guidelines ondesirable amino acid substitutions are given below as part of thesection on synthetic peptides. In addition to substituted and truncatedsequences, extended sequences can be prepared in which additional aminoacids are added at either end of a selected epitope region for thepurpose of facilitating attachment to solid phase supports andmacromolecular carriers.

As an example, useful truncated sequences of the peptide extending fromamino acid residue 502 through 541 of HIV1_(SF2) gp41 discussed aboveinclude a peptide with the sequence of amino acid residues 512-531:

GIVGAMFLGFL

GAAGSTMGA [SEQ ID NO. 12]

and also a sequence extending from amino acid residue 518 through aminoacid residue 527:

FLGFLGAAGS [SEQ ID NO. 13]

Another particularly useful truncated peptide is a truncated sequence ofthe peptide extending from amino acid 7 through 43 of gp120 of HIV2_(Nz)has the following sequence

LLIAIVLASAYLIHCKQ [SEQ ID NO. 14]

The peptide can be prepared in a wide variety of ways. The peptide,because of its relatively small size, can be synthesized in solution oron a solid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available today and can be usedin accordance with known protocols. See, for example, Stewart and Young,Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., 1984; andTam et al., J. Am Chem. Soc. (1983) 105:6442.

Alternatively, hybrid DNA technology can be employed where a syntheticgene is prepared by employing single strands which code for thepolypeptide or substantially complementary strands thereof, where thesingle strands overlap and can be brought together in an annealingmedium so as to hybridize. The hybridized strands then can be ligated toform the complete gene, and, by choice of appropriate termini, the genecan be inserted into an expression vector, many of which are readilyavailable today. See, for example, Maniatis et al., Molecular Cloning, ALaboratory Manual, CSH, Cold Spring Harbor Laboratory, 1982. Or, theregion of the viral genome coding for the peptide can be cloned byconventional recombinant DNA techniques and expressed in procaryotic oreukaryotic expression systems to produce the desired peptides.

Preferably, the immunogen will be enriched for the desired epitopes towhich antibody-producing B lymphocytes will respond by producingantibodies that will neutralize and inactivate essential steps in thelife cycle of HIV infection. As used herein, “enriched” means that adesired epitope constitutes at least 25% of the HIV protein, preferablyat least 50%, and most preferably about 95%. More particularly,solutions containing disrupted virus lysate or extracts, or supernatantof biologically-expressed recombinant proteins or disrupted expressionvectors or proteins containing mimicked epitopes can be enriched forsuch proteins, as desired, using purification methods, such as, forexample, polyacrylamide gel electrophoresis. Immunoaffinity purificationis a preferred and convenient method for purification of proteins andpeptides containing the desired HIV epitopes, e.g., affinitypurification using mono specific affinity purified polyclonal ormonoclonal antibodies. The extent to which the peptides are purifiedfrom the solutions for use as an immunogen can vary widely, i.e., fromabout 50%, typically at least 75% to 95%, desirably 95% to 99% and, mostdesirably, to absolute homogenity.

To obtain antibodies to the desired epitopes, animals are immunized witheither the peptides of interest or HIV proteins containing them whichhave been treated to remove carbohydrates and HLA antigens as disclosedabove. Immunization protocols are well known and can vary considerablyyet remain effective. See Colco, Current Protocols in Immunology, JohnWiley and Sons, Inc. 1995. The proteins and/or peptides can be suspendedor diluted in an appropriate physiological carrier for immunization.Suitable carriers are any biologically compatible, non-toxic substanceto deliver and/or enhance the immunogenicity of the peptides, includingsterile water and 0.9% saline.

Alternatively, the peptides can be coupled to a carrier molecule beforebeing used as an immunogen. One preferred technique, for example,discussed in more derail below, involves the attachment of the proteinsand fragments thereof to multiple repeats of a glycopeptide, such asmuramyl dipeptide (MDP), to form a microparticle, typically less than 1micron, and preferably less than 0.2 microns, in diameter. Themicroparticle then can be dispersed in a pharmaceutical carrier forinjection. This procedure achieves a high density of the peptide whichcan be used to elicit the desired immune response. The selection ofcarrier will vary depending upon the route of administration andresponse. The compositions can be sterilized by conventional, well-knownsterilization techniques. The peptides can be administered by oral orparenteral routes, preferably the latter.

Immunogenic amounts of antigenic preparations enriched for the desiredepitopes are injected, generally at concentrations in the range of 1 μgto 20 mg/kg body weight of host. Administration can be by injection,e.g., intramuscularly, peritoneally, subcutaneously, intravenously, etc.Administration can be one time or a plurality of times, usually at oneto four week intervals.

Immunized animals are monitored for production of antibody to thedesired epitope. High affinity complement fixing IgG antibody ispreferred for passive immunotherapy and can be used intact or asfragments such as Fv, Fab, F(ab′)2. Antibody fragments may be preferablewhen greater tissue penetration is desirable. Antibodies and fragmentscan be given alone or as conjugates with toxic substances or isotopes.Once the desired antibody response is attained, blood is collected by,for example, venipuncture, cardiac puncture, or plasmapheresis.Antibodies are purified from the complex serum or plasma mixture inaccordance with conventional procedures, including, for example, saltprecipitation, ion exchange chromatography, size chromatography,affinity chromatography. Oftentimes, a combination of methods is used.Immunoaffinity chromatography is a preferred method.

To circumvent possible antigenicity in a human receiving antibodyderived from a non-human animal, recombinant antibodies can beconstructed. One type of recombinant antibody is a chimeric antibody,wherein the antigen binding fragment of an immunoglobulin molecule(variable region) is connected by a peptide linkage to at least part ofanother protein not recognized as foreign by humans, such as theconstant portion of a human immunoglobulin molecule. This can beaccomplished by fusing the animal variable region exons with human kappaor gamma constant region exons. Various techniques are known to theskilled artisan, such as those described in PCT 86/01533, EP171496, andEP173494, the disclosures of which are incorporated herein by reference.A preferred type of recombinant antibodies is CDR-grafted antibodies.

PHARMACEUTICAL FORMULATIONS AND THEIR USE

The antibodies of this invention that neutralize infectivity, killinfected CD4 lymphocytes and inactivate functionally important events inthe life cycle of HIV are incorporated as components of pharmaceuticalcompositions. The compositions comprise a Therapeutic or prophylacticamount of at least one of the antibodies of this invention, anddesirably an antibody cocktail, with a pharmaceutically acceptablecarrier. A pharmaceutically acceptable carrier is any compatible,non-toxic substance suitable for the delivery of the antibodies to thepatient. Thus, this invention provides compositions for parenteraladministration which comprise a solution of antibody dissolved in anacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., water, buffered water, 0.4% saline, 0.9%saline, 0.3% glycine and the like. These solutions are sterile andgenerally free of particulate matter. The compositions further cancomprise pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, toxicity adjusting agents and the like. For example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate, etc., can be used. The concentration of antibody in theseformulations can vary, typically from less than about 0.1 mg/ml to asmuch as 150 or 200 mg/ml, preferably between about 1 mg/ml and about 20mg/ml, and will be selected primarily based on fluid volumes,viscosities, etc., preferably for the particular mode of administrationselected. Determining the concentration of a particular antibody orantibody cocktail is within the abilities of one of ordinary skill inthe art. Thus, a typical pharmaceutical composition for intravenousinfusion can be made up to contain 250 ml of sterile Ringer's solutionand 100-200 mg of antibody. Compositions for intramuscular injection canbe made up to contain 1 ml sterile buffered water and about 20 to about50 mg of antibody. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example, Remington'sPharmaceutical Science, 15th Ed., Mack Publishing Company, Easton, Pa.(1980), which is incorporated herein by reference. Such compositions cancontain a single antibody which is, for example, specific for certainstrains of HIV or for a single protein or glycoprotein expressed by mostand, more preferably, all strains of HIV. Alternatively, apharmaceutical composition can contain more than one antibody to form a“cocktail.” For example, a cocktail containing antibodies againstvarious proteins and strains of HIV would be a universal product withtherapeutic or prophylactic activity against the great majority of theclinical isolates of HIV. The cocktail can contain antibodies which bindto epitopes on proteins or glycoproteins of the HIV envelope, forexample, or can contain a combination of antibodies to epitope sitesidentified above on HIV1 _(SF2) Env proteins gp160, gp120, and gp41; Gagprotein p7, p17 and p24; reverse transcriptase heterodimer p66/55 andprotease p10, or a subgroup thereof, thus neutralizing a series ofepitopes crucial in the life cycle of HIV. Antibodies to epitope siteson other neutralizing or inactivating regions of HIV proteins also, ofcourse, can be employed.

For example, antibodies which modify attachment, cell entry,transcription, translation, assembly, targeting of the mature virion tothe plasma membrane and extrusion of the virion will interfere with HIVlife cycle events. Antibody cocktails will more frequently be employedto obtain inactivation of multiple essential HIV proteins. This will beof therapeutic benefit in particular within virions lacking the outerenvelope but possibly are infectious should they gain cell entry byother mechanisms such as micropinocytosis or transfection or the like.The molar ratio of the various antibody components usually will notdiffer by more than a factor of 10, more usually by not more than afactor of 5, and will usually be in a molar ratio of about 1:1-3 to eachof the other. antibody components.

With respect to antibodies to the nine specific peptides set forthabove, a desirable antibody cocktail comprises antibodies to the twoenvelope gp120 peptides and gp41 peptide. More desirably, the cocktailcomprises antibodies to those three epitope regions plus an antibody tothe protease p10 epitope region. Even more desirably, the cocktailcomprises antibodies to those four epitope regions plus antibodies to ateast one of the other five enumerated epitope regions. in a mostpreferred embodiment, the cocktail comprises antibodies to all nine ofthe epitope regions.

The antibodies and antibody cocktails of the present invention can beadministered independently or given in conjunction with otheranti-retroviral agents. The current status of the development of otheranti-retroviral agents, and of anti-HIV agents in particular, isreviewed in Mitsuya et al., Nature 325:773-778, 1987.

The antibodies and peptides of this invention can be stored in liquidformat at various temperatures known to preserve antibody activity, e.g.−70° C., −40° C., −20° C., and 0-4° C. or lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immune globulins, purifiedantibodies, and immunogens composed of proteins, glycoproteins, andpeptides. Art-known lyophilization and reconstitution techniques can beemployed and it will be appreciated by those skilled in the art thatlyophilization and reconstitution can lead to varying degrees ofantibody activity loss (e.g., with conventional immune globulins, IgMantibodies tend to have greater activity loss than IgG antibodies) andthat doses may have to be adjusted to compensate for any loss.

The compositions containing the present antibodies or cocktails thereofcan be administered for the therapeutic and/or prophylactic treatment ofHIV infections. In therapeutic application, compositions areadministered to a patient already infected with HIV, in an amountsufficient to treat or at least partially arrest the infection and itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the infection and the general state of thepatient's own immune system, but generally range from about 0.1 to about200 mg of antibody per kilogram of body weight with dosages of from 0.5to 25 mg per kilogram being preferred. The compositions of thisinvention can be employed in serious disease states that arelife-threatening or potentially life-threatening situations. In suchcases, it is possible and may be felt desirable by the treatingphysician to administer substantial excesses of these antibodies.

In prophylactic applications, compositions containing the presentantibodies or a cocktail thereof are administered to a patient notalready infected by HIV, but perhaps recently exposed to or thought tohave been exposed to, or at risk of being exposed to The virus (such as,for example, the newborn of an HIV infected individual), or immediatelyfollowing an exposure or suspected exposure to HIV. If the compositionis to be administered to an HIV-infected pregnant female, it can begiven once or multiple times prior to delivery to reduce HIV infectivityin maternal blood and thereby reduce the risk of HIV transmission to thenewborn. The newborn at risk also can be treated to further reduce therisk of contracting HIV. An amount defined to be a “prophylacticallyeffective dose” generally ranges from 0.1 mg to 25 mg per kilogram ofbody weight, depending upon the patient's state of health and generallevel of immunity.

In addition, the antibodies of the present invention can find use as atarget-specific carrier molecule. An antibody can be bound to a toxin toform an immunotoxin or a radioactive material or drug to form aradiopharmaceutical or pharmaceutical. Methods for producingimmunotoxins and radiopharmaceuticals are well known (see, for example,Cancer Treatment Reports 68:317 (1984)). Heteroaggregates of antibodiesof the present invention and human T-cell activators, such as monoclonalantibodies to the CD3 antigen or to the Fc gamma receptor on T-cells,can enable human T-cells or Fc-gamma bearing cells (such as K cells orneutrophils) to kill HIV infected cells via antibody dependentcell-mediated cytolysis (ADCC). Such heteroaggregates can be assembled,for example, by covalently cross-linking the anti-HIV antibodies to theanti-CD3 antibodies using the heterobifunctional reagentN-succinimidyl-3-(2-pyridyl dithiol)propionate, as described inKarpowsky et al., J. Exp. Med. 160:1686 (1984, which is incorporated byreference herein.

Other anti-HIV agents also can be included in the formulations, such as3′-azido-3′-deoxythymidine, 2′,3′-dideoxycytidine, 2′,3′-dideoxy-2′,3′-didehydrocytidine, etc.

In addition to antibody compositions, compositions comprising thepeptides of this invention can be administered for therapeutic andprophylactic vaccination of HIV-infected individuals. For therapeuticapplication, compositions comprising peptides, either as isolatedpeptides optionally modified as discussed above or contained within HIVproteins treated as described above and desirably coupled to an MDPmicroparticle to further stimulate immunogenicity, are administered to apatient infected with HIV. The amount of peptide administered is chosenso as to stimulate antibody production to functional HIV epitopes notpreviously recognized by the patient's immune system so that thestimulated antibodies can arrest the infection. In prophylacticapplications, compositions of the peptides coupled to the microparticleMDP are administered to persons not infected with HIV to stimulate theproduction of antibodies against otherwise unrecognized epitopes toprovide a protective function against future infection.

DIAGNOSTIC AND PROGNOSTIC USES OF ANTIBODIES AND ANTIGEN

The antibodies and epitopes recognized by them and disclosed in thepresent invention also are useful for the diagnosis and management ofHIV infection. Typically, diagnostic assays employing antibodies and/ortheir respective antigens entail the detection of the antigen-antibodycomplex. Numerous immunoassay configurations have been described andemploy either labeled or unlabeled immunochemicals for this purpose.When unlabeled, the antibodies find use, for example, in agglutinationassays, antibody dependent complement mediated cytolysis assays, andneutralization assays. Unlabeled antibodies can be used in combinationwith other, labeled, antibodies (second antibodies) that are reactivewith the primary antibody, such as antibodies specific forimmunoglobulin. Unlabeled antibodies can be used in combination with alabeled antibody which is reactive with a non-competitive epitope on thesame antigen, such as in sandwich type immunoassays, or in combinationwith a labeled antigen. Alternatively, the antibodies can be directlylabeled and used in both competitive and non-competitive immunoassays.These assay types and configurations are well known in the art. A widevariety of labels can be employed, such as radioisotopes, fluorescenttags, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,ligands (particularly happens), etc. Numerous types of immunoassays areavailable and, by way of example, include those described in U.S. Pat.Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876.

Commonly, the antibodies and peptides of the present invention areutilized in enzyme immunoassays, where, for example, the subjectantibodies, or their respective antigens are conjugated to an enzyme andthe immunoassay is configured to provide maximal sensitivity andspecificity in detecting HIV antigens in biological samples such ashuman blood serum, saliva, semen, vaginal secretions or viral infectedcell culture suspension.

Kits also can be designed for use with the subject antibodies for use inthe detection of HIV infection or the presence of HIV antigen. The kitscomprise antibodies of the present invention optionally in conjunctionwith additional antibodies specific for other epitopes of HIV. Theantibodies, which can be conjugated to a label, unconjugated or bound toa solid support such as the surface of a microtiter plate well or apolystyrene bead, are included in the kits with buffers, such as Tris,phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g.,bovine serum albumin, or the like. Generally, these materials will bepresent in less than about 5% wt. based on the amount of activeantibody, and usually present in total amount of at least about 0.001%wt. based again on the antibody concentration. Frequently, it will bedesirable to include an inert extender or excipient to dilute the activeingredients, where the excipient can be present in from about 1% to 99%wt. of the total composition. Where a second antibody capable of bindingto the antibody is employed, the second antibody usually will be presentin a separate vial. The second antibody typically is conjugated to alabel and formulated in an analogous manner with the antibodyformulations described above. The subject epitope recognized by theantibody can be provided labeled or non-labeled and can be provided aspart of a larger protein (synthetic, recombinant, or native), with orwithout modification such as the addition of spacer arms, amino groups,or cysteine residues, which can be used to attach the peptide to asupport and extend it from the surface of the support. Suchmodifications are employed to provide the epitope in an arrangement tooptimize immunoreactivity with the antibody. Such peptides areformulated in a manner analogous to that of the epitope-containingproteins as described above.

The detection of HIV antigens, or the whole virus, in various biologicalsamples is useful in diagnosing a current infection by HIV, evaluatingresponse to therapy, enumerating infected cells, serotyping HIV strains(clades), identifying and quantitating virulence factors associated withprimary infection, progression and complications such as peripheralneuropathy, multi focal leukoencephalopathy, and Kaposi's sarcoma.Biological samples can include, but are not limited to, blood, serum,saliva, semen, tissue biopsy samples (brain, skin, lymph nodes, spleen,etc.), cell culture supernatants, disrupted eukaryotic and bacterialexpression systems, and the like. Presence of virus, viral antigens,virulence factors, and serotyping determinants are tested for byincubating the antibody with the biological sample under conditionsconducive to immune complex formation, followed by the detection ofcomplex formation. In one embodiment, complex formation is detectedthrough use of a second antibody capable of binding to the primaryantibody and typically conjugated to a label. The second antibody isformulated in a manner analogous to that described for the primaryantibody formulations described above. In another embodiment, theantibody is attached to a solid phase support which then is contactedwith a biological sample. Following an incubation step, labeled antibodyis added to detect the bound antigen. In another embodiment, theantibody is conjugated to a detection label and following an incubationstep with a biological sample, such as cells or tissue sections, thesample is evaluated by flow cytometry or microscopy for the antigen.

PREPARATION AND USE OF SYNTHETIC PEPTIDES

Peptides of this invention can be modified by introducing amino acidsubstitutions into the peptide. Substitutions may be desirable to varyone or more particular amino acids to more effectively mimic theepitopes of the different retroviral strains or to enhance immunologicalresponses or MHC interactions with the epitope resulting in greaterimmunogenicity of the mimicked epitope when used for immunization orvaccination. In addition, it can be desirable to make certain amino acidsubstitutions to enhance the chemical stability of the peptide.

More specifically, a polypeptide employed in the subject invention neednot be identical to any particular HIV polypeptide sequence, so long asit is able to provide immunological competition with proteins of atleast one of the strains of HIV. Therefore, the subject polypeptides canbe subject to various changes, such as insertions, deletions, andsubstitutions, either conservative or non-conservative, where suchchanges will enhance the desired activity of the peptide. Conservativesubstitutions are substitutions with similar amino acids within a groupsuch as neutral, acidic, basic, and hydrophobic amino acids. Examples ofsubstitutions within such groups would include gly, ala; val, ile, leu;asp, glu; asn, gin; ser, thr; lys, arg; phe, tyr; and nor, met.Additional amino acid substitutions, obtained by application ofmolecular modelling software to HLA allotyping database classification(DNA and serological), are shown:

Group, 1 letter code Group, 3-letter code F, S phe, ser Q, R gln, arg K,N lys, asn E.G glu, gly G, R gly, arg H, Q his, gln I, T ile, thr V, Aval, ala D, N asp, asn Y, F tyr, phe I, F ile, phe K, E lys, glu E, L, Rglu, leu, arg Y, L tyr, leu S, V ser, val Y, N, F, D tyr, asn, phe, aspD, R asp, arg L, F leu, phe

In a preferred embodiment of the invention, amino acid modifications aremade so as to substitute hydrophilic residues on the more hydrophilicend of the peptide of interest and hydrophobic residues on the morehydrophobic end of the peptide. Such substitutions result in theformation of an amphipathic helix with the desired epitope bracketedbetween the substitutions. Substituted amino acids in the D isomer canbe employed to bracket epitopes to protect and stabilize the epitope andenhance immunogenicity of the epitope. Since D-amino acids are notcleaved by intracellular enzymes, such protion provides peptide epitopesof the desired length for interaction with MHC molecules when they areinserted at appropriate sites. This is described in detail in Example8.5 below.

Usually, the modified sequence will not differ by more than about 20%from the sequence of at least one strain of the human immunodeficiencyretrovirus except where additional amino acids are added at one or bothtermini for the purpose of providing an “arm” by which the peptide ofthis invention conveniently can be immobilized on solid phase supports,attached to macro molecules or modified to enhance immunogenicity byaltering or enhancing MHC binding and presentation. The arms cancomprise a single amino acid or as many as 50 or more amino acids, andtypically are 1 to 10 amino acids, in length.

Amino acids such as tyrosine, cysteine, lysine, glutamic or asparticacid, or the like can be introduced at the C-or N-terminus of thepeptide or oligopeptide to provide for a useful functionality forlinking. Cysteine is particularly preferred to facilitate covalentcoupling to other peptides or to form polymers by oxidation.

Additionally, the peptide or oligopeptide sequences can differ from thenatural sequence by the sequence being modified by terminal-NH₂acylation (e.g., acetylation), thioglycolic acid amidation, or terminalcarboxy amidation (e.g., with ammonia or methylamine)-, to providestability, increased hydrophobicity for linking or binding to a supportor other molecule, or for polymerization.

Thus, for example, in a preferred embodiment of the peptides disclosedherein, one or more cysteine residues or a combination of one or morecysteine residues with spacer amino acids can be added to the termini ofthe peptide. Glycine is a particularly preferred spacer when individualpeptides are desired. When multiple peptide repeats of the peptide aredesired, the peptide is synthesized off of a lysine core to form atetravalent peptide repeat. The configuration is shown by way ofexample. Preferred peptides for use in oxidative polymerization arethose in which at least two cysteine residues are added to the terminiof a desired peptide. When two cysteine residues are present at the sameend of the peptide, a preferred embodiment exists when the cysteineresidues are separated by one to three spacer amino acid residues,preferably glycine. The presence of cysteine residues may allow theformation of dimers of the peptide and/or increase the hydrophobicity ofthe resulting peptide which facilitates immobilization of the peptide insolid phase or immobilized assay systems. Of particular interest is theuse of the mercapto group of cysteines or thioglycolic acids used foracylating terminal amino groups or as the first amino acid for buildingmultiple peptide repeats or the like for linking two of the peptides oroligopeptides or combinations thereof by a disulfide linkage or a longerlinkage to form polymers that contain a number of epitopes. Suchpolymers have the advantage of increased immunological reaction. Wheredifferent peptides are desired for immunization, they are individuallyassembled and combined in a cocktail to provide the additional abilityto induce antibodies what immunoreact with several antigenicdeterminants of different HIV isolates. To achieve the formation ofantigenic polymers (synthetic multimers), compounds can be employedhaving bis-haloacetyl groups, nitroarylhalides, or the like, where thereagents are specific for these groups. The linking between the one ortwo mercapto groups of the peptides or oligopeptides can be a singlebond or a linking group of at least 2 or more carbon atoms.

LINKING PEPTIDES TO MACROMOLECULAR CARRIERS

The subject peptide can be employed linked to a soluble macromolecular(e.g., not less than 5 kDal) carrier. Conveniently, the carrier can be apoly(amino acid), either naturally occurring or synthetic, to whichantibodies are unlikely to be encountered in human serum. Examples ofsuch carriers are poly-L-lysine, keyhole limpet hemocyanin,thyroglobulin, albumins, such as bovine serum albumin, tetanus toxoid,etc. The choice of the carrier is primarily dependent upon the ultimateuse intended for the antigen and one of convenience and availability. Ina preferred embodiment, the carrier comprises multiple repeats ofglycopeptide a microparticle which can be synthesized or isolated fromcertain bacteria such as Propionibacterium acini or the like. Thismicroparticle is composed of muramyl dipeptide extensively crosslinkedresulting in multimeric configurations.

When muramyl dipeptide is isolated from Propionibacterium acini orrelated organisms, strain selection is helpful, and selection is basedon chemical analysis of the bacterial cell wall. The preferredembodiment is muramyl dipeptide extensively crosslinked with a dipeptidecomposed of L-alanine-D-isoglutamine.

From preliminary experiments, strain differences have been identified inwhich dipeptide composition and peptide length vary. Isolates with highconcentrations of lipid A and O-acylated beta myristate are componentsof the cell wall. Preliminary experiments showed these differences areassociated with increases in toxicity and decreases in adjuvant effect.Strain selection and the purification of the preferred embodiment isdiscussed by way of Example 4, below.

The MDP microparticle can be synthesized by employing procedures knownin the art. It has been well established that MDP is a potentimmunostimulant but has significant toxicity. Many attempts to reduceMDP toxicity have employed procedures to delay release, such as MDPincorporation into liposomes or other related compounds or modificationof terminal groups. Chemical modification resulted in marked reductionin the desired adjuvant effect, and designs which change delivery ratehave been difficult to control. By way of example, MDP microparticleconfiguration, size parameters, and antigen delivery attachment methodsare provided below. Removal of lipids from the microparticleconfiguration facilitates rapid internalization of MDP by antigenpresenting cells (APC). Antigen presenting cells are predominantly ofmonocytes lineage and include monocytes, macrophage, Histiocytes, Kuffercells, Dendritic cells, Langerhans cells, etc. and participate inantigen processing and antigen presentation through MHC associatedevents. Factors which contribute to the development of immune responsesto foreign protein can be in part, determined by amino acid sequence andsequence susceptible to protease cleavage in the micro environment.Successful immune responses are most frequently observed to peptideswhich form an amphipathic helix with a hydrophobic terminus, preferablyon the amino terminal end, and hydrophilic amino acids most frequentlyon the carboxyl terminal end. Sequence configurations that are resistantto protease degradation and form amphipathic helix arrangements arefrequently strong immunogens. Residues which contain praline in thesequence are generally poorly immunogenic by preventing helix formationand glycosylation sites are less favorable and frequently inhibitresponses directed at peptide epitopes. Antigen challenge which resultsin a successful immunological response in the host animal requiresantigen processing and presentation of antigen through MHC associatedevents. Exogenous antigen is primarily processed by antigen presentingcells (APC) after internalization into endosomes. Following proteolysisby enzymes, such as cathepsin D, which are present and react in thisacid environment, peptide fragments which satisfy the criteria describedabove are assembled with MHC class II and presented on the cell surface.When peptides are presented in sufficient density immune events result.The type of immune response is driven by the density of peptide per APC,micro-environment, the cytokine environment, and the lymphocyte typeinitially stimulated by antigen presenting cells. Followinginternalization, a cascade of cytokine responses is induced whichmodifies the micro-environment and establishes conditions conducive ofimmunological events.

By way of example, a unique MDP microparticle (0.01-0.2 micron) is usedto deliver immunogen to antigen presenting cells resulting in immuneresponses to poorly immunogenic epitopes not observed using conventionalmethods as shown in Example 5 below. Quantitation of these immuneresponses demonstrate 10 to 100 fold increases in antibody concentrationas compared to other adjuvants.

Subject peptides employed as immunogen can be linked to the carboxylterminal amino acid moiety of muramyl dipeptide using either the aminoor carboxyl terminus of the subject peptide or to the aldehyde oxidationproduct of the carbohydrate moiety as disclosed in the examples. Therewill be at least one molecule of the subject peptide per MDPmicroparticle, preferably 10-100 molecules of subject peptide per MDPmicroparticle and most preferably 100 to 1000 subject peptides per MDPmicroparticle. Carrier size and available linkage groups, therefore,will influence the number of subject peptides per carrier.

Macro-carrier composition affects immunogenicity by influencingpreferential cell uptake, peptide half-life, and antigen presentationthrough MHC immunological events. One or more different subject peptidescan be linked to the same macro-carrier but preferably a single subjectpeptide is attached either in the univalent or tetravalent configurationto the macro-carrier. When immunization with more than one subjectpeptide is desired, a cocktail of subject peptide macro-carrierconjugates can be prepared by mixing individual conjugates at ratios tooptimize immunogenicity of each subject peptide introduced in thecocktail. In this configuration sufficient peptide is available on eachmacro-carrier conjugate (100-1000 peptides) to enhance antigenpresentation by a single antigen presenting cell. Immunogenicity of thesubject peptide will be optimized by adjusting both the number ofsubject peptides per macromolecular carrier, presentation configuration,such as amino versus carboxyl attachment, terminal amino acidmodification, and space arm length and composition, as disclosed. Inthis configuration, antigen processing by the antigen presenting cellresults in a high density, usually more than 100 and most frequentlymore than 500 peptides, presented at the cell surface of the antigenpresenting cell through MHC interactions. With this configurationsignificantly higher concentrations of antibody are produced, followingimmunization, as shown in the examples below.

The manner of linking is conventional, employing such reagents asp-maleimidobenzoic acid, p-methyldithiobenzoic acid, maleic acidanhydride, succinic acid anhydride, glutaraldehyde, etc. The linkage canbe made at the N-terminus, C-terminus, or at a site intermediate to theends of the molecule. With multiple repeats of muramyl dipeptide,attachment of the subject peptide to aldehyde groups produced by themild oxidation of sugar residues with, for example, sodium periodatefollowing mild reduction with sodium borohydride and the like, theSchiff's base intermediate is converted to a stable covalent linkage.The number of peptides per microparticle can be controlled by varyingoxidation conditions and quantitated by employing a radioactive tracer.These methods are well known in the art. The method of attachment andattachment configuration can vary from peptide to peptide as needed toachieve the desired response.

Various assay protocols familiar to those skilled in the art can beemployed for detecting the presence of either antibodies to retroviralprotein epitopes or detecting retroviral proteins in complex proteinmixtures. Of particular interest is a novel assay herein disclosed inwhich the subject peptide is covalently attached to a detection labelsuch as horseradish peroxidase and the native HIV protein expressingepitope or epitopes is either directly or indirectly attached to a solidphase support. In this configuration an antibody which recognizes thepeptide epitope will bridge the epitope on the solid phase with theepitope on the label. With this method the epitope reactivity of anantibody to HIV can be determined and quantitated by varying thepeptides attached to the label. Peptide epitopes which are associatedwith HIV serotype, virulence factors or other HIV characteristics can beidentified and measured in any sample expressing these epitopes by a onestep competitive immunoassay herein disclosed and provided by way ofexample.

Use of Antibodies and their Respective Epitopes in ImmunoaffinityPurification Procedures

Antibodies specific for epitopes contained within HIV proteins andpurified proteins containing these epitopes are of particular advantagefor use in immunoaffinity purification of proteins and peptidescontaining these epitopes and antibodies reactive with them. Generally,the antibodies will have affinity association constants on the order of10⁸ to 10¹² M. Such antibodies can be used to purify proteins andpeptides containing the epitopes of interest. Oftentimes, geneticallymodified bacteria can be used to make HIV proteins, and the recombinantfusion proteins of interest can be purified from the culture medium ofthe recombinant expression system if the expressed protein is secreted,or from the components of the disrupted biological expression system ifit is not secreted, or from complex biological mixtures of proteins ofwhich some or one component contains the epitope mimicking an epitope onHIV. Generally, the antibodies which are capable of reacting with HIVepitopes are attached to or immobilized on a substrate or support. Thesolution containing the epitopes then is contacted with the immobilizedantibody under conditions suitable for the formation of immune complexesbetween the antibody and the protein containing the epitope. Unboundmaterial is separated from the bound immune complexes, and the boundproteins are released from the immobilized antibody and recovered in theeluate.

Similarly, proteins or peptides containing epitopes of HIV or mimickingepitopes of HIV can be attached to or immobilized on a substrate orsupport and used to isolate antibodies of interest from a solution. Asolution containing the antibodies, such as plasma from which albuminhas been removed, is passed through a column of immobilized peptides orproteins containing the desired epitopes and, following immune complexformation, non-reactive antibody is separated from the bound immunecomplex and the antibody is released with an elution buffer andrecovered in the eluate. This is of particular value in purifyingprotein containing epitopes mimicking epitopes of HIV but derived fromsources phylogenetically unrelated to HIV.

Typically, antibodies are crudely purified from hyperimmune sera.Ascites fluid or cell culture supernatants and proteins or peptidescontaining epitopes mimicking HIV epitopes will be crudely purified frombiological sources such as, but not limited to, body fluids, blood,blood components, cell extracts, tissue extracts of both adult andembryonic origins, and culture supernatants, extracts of cultured cells,venoms, and recombinant fusion products prior to attachment to asupport. Such procedures are well known by those skilled in the art andmay include fractionation with neutral salts at high concentration.Other purification methods, such as ion exchange chromatography, gelfiltration chromatography, preparative gel electrophoresis, or affinitychromatography, also can be used to increase the purity of thepreparation prior to its use as an immunoabsorbant. Affinity purifiedantibody can be prepared when desired by reacting crudely purifiedantibody preparations with a support matrix to which the reactiveepitope or protein containing the epitope has been attached.

Of particular interest are antibodies to HIV epitopes phylogeneticallymimicked through nature. Such antibodies are useful as therapeuticagents and also are useful for studying HIV by allowing purification ofHIV proteins and the mapping of HIV proteins for sequence location andfunction. Such antibodies can be produced by immunization with HIVproteins/peptides derived from HIV and immunoaffinity purified byreacting the resultant hyperimmune polyclonal multivalent antisera withproteins/peptides derived from non-HIV sources such as embryonicproteins, venoms, and non-HIV microbial/viral components immobilized ona support as discussed above. The resulting immunoaffinity purifiedantibody is epitope specific for an epitope or epitopes shared by HIVand the phylogenetically unrelated protein used for itsimmunopurification. These epitope specific antibodies have particularutility in the immuno-affinity purification of proteins and peptides ofboth HIV and non-HIV origin. Such antibodies can be used to map thelocation of the epitope on HIV to determine its sequence, evaluatefunctional importance in the life cycle of HIV, its distribution withinthe clades of HIV and among other retroviridae, its association with HIVvirulence, and, when non-toxic to man but neutralizing a crucialfunction in the life cycle of HIV, used to treat HIV infection.

The support to which the antibodies or epitopes are immobilizeddesirably has the following general characteristics: (a) weakinteractions with proteins in general to minimize non-specific binding,(b) good flow characteristics which allow the flow through of highmolecular weight materials, (c) possession of chemical groups that canbe activated or modified to allow chemical linkage of the antibody orepitope, (d) physical and chemical stability in the conditions used tolink the antibody, and (e) stability to the conditions and constituentsof the buffers required for absorption and elution of the antigen. Somesupports commonly used are agarose, derivatized polystyrenes,polysaccharides, polyacrylamide beads, activated cellulose, glass andthe like. Various chemical methods exist for the attachment ofantibodies and antigens to substrate supports. See generally,Cuatrecasas, P., Advances in Enzymology 36:29 (1972). The antibodies andantigens of the present invention can be attached directly to thesupport or, alternatively, through a linker or spacer arm. Generalconditions required for immobilization of antibody and antigens tochromatographic supports are well known in the art. See, for example,Tijssen, P., 1985, Practice and Theory of Enzyme Immunoassay, which isincorporated herein by reference. Actual coupling procedures will dependslightly on the characteristics and type of the antibody or the antigento be coupled. Attachment typically occurs through covalent bonds.

An immune serum, ascites fluid or culture supernatant rich in antibodyor extract or lysate of HIV virus, the supernatant or extract from acultured biological expression system, the supernatant or extract from asuspension of the disrupted cells tissue or blood component (adult andembryonic) or other complex protein mixtures such as venoms, bodyfluids, or culture products containing the epitope then is added to theappropriate separation matrix. The mixture is incubated under conditionsand for a time sufficient for antigen-antibody attachment to occur,usually at least 30 minutes, more usually 2 to 24 hours. The immobilizedimmune complexes containing the specifically bound antibody or epitopesthen are separated from the complex mixture and extensively washed withabsorption buffer to remove non-bound contaminants. The immune complexesthen can be dissociated with an elution buffer compatible with theparticular support, the attached protein, and the eluate protein. Theelutable protein, antigen, or antibody is recovered in the eluate.Elution buffers and techniques are well known by those skilled in theart. Peptides that contain the epitope recognized by the antibody can beused in the elution buffer to compete for the antibody binding site andelutions can be performed under mild elution conditions. The selectivelyabsorbed protein can be eluted from the affinity absorbent by alteringthe pH and/or ionic strength of the buffer or with chaotropic agents.The selection of an elution buffer, its concentration and other elutingconditions are dependent on the Characteristics of the antibody-antigeninteraction, and once determined should not be subject to significantchange.

The eluted protein may require adjustment to a physiologic pH and ionicstrength if low or high pH or ionic strength buffers or chaotrophicagents are used to dissociate the immune complex. Such adjustment can bemade by dialysis or gel filtration chromatography. These methods alsopermit the eluted protein to regain its native conformation.

The foregoing methods yield, e.g., substantially purified proteinscontaining epitopes of, or mimicking epitopes of, HIV and antibodiesreactive with the epitopes. The purified proteins typically will begreater than 50% pure, more usually at least 75% pure, and frequentlygreater than 95% to 99% pure.

Other features and advantages of the present invention will becomeapparent from the following experimental descriptions, which describethe invention by way of example. The examples further illustrate theprocess of this invention but are not meant to limit the invention inany way.

EXAMPLE 1 Preparation of Human Anti-HIV Antibody (IgG fraction) PoolsFor Use In Mapping HIV Epitope Differences Between Man and Goat ImmuneReactions

Human sera from HIV-infected patients were obtained from a communityhealth clinic with patient permission, patient signed informed consent,physician approval and local IRB approval. All sera were initiallyscreened for HIV reactivity at a dilution of 1:10 employing acommercially available test kit from Abbott Laboratories. Sera with highreactivity (arbitrarily defined by test result absorbences greaterthan 1) were further evaluated by Western Blot analysis to identifythose sera with antibody reactivities-to most HIV proteins (env, gag andpol). Patient sera that demonstrated good reactivity to most HIVproteins as defined were further evaluated by microcultureneutralization assays to identify the sera-containing antibodyspecifities which would neutralize HIV infectivity in microcultureassays (total culturable infectious dose (TC1D) neutralization).Twenty-nine patients' sera with high antibody titer reactivity to gp160,gp120, p66/55, gp41, p24, and p17 and p10, and neutralized HIVinfectivity in microculture were identified and pooled (20-40 ml each).Further evaluation of the pooled anti-human HIV revealed broadneutralization activity against multiple strains of HIV and high titerantibody (positive by Western Blot 1:100 or greater) to the nine epitoperegions described in detail above. Human IgG was purified from thisserum pool employing conventional procedures. Following purification thetotal IgG concentration was adjusted to 10 mg/ml, its composition andpurity was evaluated by standard immunoassay procedures. The resultsdemonstrated a purity of greater than 98% and a composition of humanIgG. The purified human anti-HIV was divided into aliquots and frozenfor subsequent use in experiments and procedures disclosed below. Thoseskilled in the art will be familiar with methods for characterizing bothantigen and antibody pools to define specificities which may be used insubsequent determinations against unknown antibodies or antigens forcomparative purposes.

As described in the following examples, this human anti-HIV was employedto follow the purification of HIV proteins from crude viral lysates andmap HIV epitopes recognized by the human immune system and incompetitive EIA to identify HIV epitopes targeted by antibodies producedin goats but not man. Western Blot analysis was employed to characterizeantibody responses in goats immunized with either purified HIV proteinscontaining peptides of interest or synthetic versions of those peptides,and to evaluate the efficacy of a microparticle carrier complex designedto amplify immune responses to poorly immunogenic peptides. Antibodieswhich neutralized HIV infectivity were evaluated by a microcultureprocedure which employed purified human CD4 lymphocytes isolated fromperipheral blood mononuclear cells (PBMC), employing standard techniqueswhich use monoclonal antibody-conjugated magnetic particles to removeunwanted cells. CD4 lymphocytes were stimulated with mitogen to increasetheir susceptibility to HIV infection and were used throughout unlessotherwise noted as the host target for HIV infection. HIV1_(SF2) wasemployed throughout unless otherwise noted as the reference HIV strain.The effect of antibody on HIV infectivity was determined by microcultureusing techniques familiar to those skilled in the art. Infectivity isexpressed as infectious units (IU). Antibody mediated reductions in IUwere associated with neutralization of virus infectivity and expressedas change in infectious unit. The human anti-HIV pool was usedthroughout all experiments described herein that required human anti-HIVantibody. This human IgG anti-HIV pool was compared to severalcommercially available human anti-HIV preparations and had equal orgreater HIV neutralizing activity and, when compared by Western Blotanalysis, was significantly more reactive.

EXAMPLE 2 Characterization of Commercially Available HIV Viral LysatesEmployed Herein

Preliminary Studies

HIV1_(MN), HIV1_(BAL) and HIV2_(NZ) were purchased from AdvancedBio-Technology, Inc., Columbia, Md., in the form of purified virallysates. Analysis of these purified viral lysates demonstrated lot tolot variation in total protein content with a range of 0.8 mg/ml to 1.2mg/ml. The protein composition of each HIV lysate was evaluated bySDS-PAGE and Western Blot analysis with human IgG anti-HIV. The HIVlysates were treated with protease inhibitors and nonionic detergents(1.0% v/v), such as Nonidet P-40 or Igepal CA630, to fully dissociateHIV proteins and glycoproteins into their monomeric forms, and clarifiedby filtration through a 0.22 micron filter. Lipids were removed withSeroClear employing standard procedures. It is well known to thoseskilled in the art that HIV incorporates human proteins into itsenvelope as part of the budding process. Such contaminants onceidentified were removed by immunoaffinity chromatography. In the initialpurification step serum contaminants present in growth media added tofacilitate cell growth and contaminants in the HIV lysate were removedby immunoaffinity chromatography employing anti-normal human serumSepharose CL6B (2-3mg antibody/gram Sepharose). Chromatography of HIVlysates were conducted at a matrix to lysate ratio of 1:1 volume/volumeat a flow rate of 10 ml/hour. The chromatography and elution weremonitored spectrophotometrically at a wave length of 280 nm. The proteinrich non-binding fraction containing the HIV related proteins wasconcentrated to 1 mg protein/ml and stored at −70° C. for future use asneeded. Proteins bound by the affinity matrices were eluted withglycine-HCl buffer pH 2.2 in 0.9% NaCl. Eluates were neutralized,dialyzed in PBS pH 7.8 containing 0.1% Igepal CA630, concentrated andstored at −70° C. for future analysis. SDS-PAGE with Western Blotanalysis of purified HIV preparations consistently demonstrated thepresence of gp160, gp120, p66/55, gp41, p10, p24, p17 and p7, employinghuman anti-HIV IgG. HIV proteins were not detected in glycine HCl eluateemploying Western Blot analysis, but SDS-PAGE gels stained withcoomassie brilliant blue for protein visualization demonstrated 2-3weakly stained bands.

Characterization of Anti-HIV Antibody Produced to Partially Purified HIVLysates

Goats (n=2) were immunized with the purified HIV proteins obtained aboveand responded immunologically with antibodies which reacted withimmunodominant epitores on HIV. Further evaluation of this antiserumdemonstrated the presence of cytotoxic antibodies which reacted withboth HIV-infected and non-infected CD4⁺ lymphocytes, and red blood cell(RBC) agglutinins were detected. These agglutinins were reacted with allhuman RBC blood groups and RBC's from rabbits and guinea pigs. Twopossibilities for these unwanted antibody specificities were considered.One possibility was contamination from HIV lysates with proteins of cellculture origin, and the second was mimicry between HIVproteins/glycoproteins and glycoproteins found in man and other animalspecies. It is well known that host membrane proteins are oftenidentified in the envelope of HIV. The incorporation of host membranecomponents into the envelope of HIV is thought to be non-specific andassociated with the budding of the mature virion. Two such proteinspreviously identified in mature virion envelope are human HLA class Iand class II antigens. Both HLA class I and class II antigens werequantitated employing an enzyme linked immunoassay and resultsdemonstrated the presence of both HLA class I and class II antigens inthese HIV preparations and at concentrations disproportionate to theirconcentration measured in cell membrane preparations derived fromuninfected culture cells. Further studies confirmed the presence of HLAclass I and class II antigens in different preparations (n=17) of HIVviral lysate. The measured concentration in these preparations wasvariable but consistently 10 to 100 times greater than that measured inmembrane extracts from uninfected control cells. The goat anti-HIVantibody was tested by Western Blot Analysis against known HLA class Iand class II isolates and confirmed to contain antibody specificitiesdirected against HLA class I, HLA class II (alpha and beta chain) andbeta 2 microglobulin. This antibody was evaluated for HLA allotypespecificity. Commercial trays containing lymphocytes of known allotypeswere employed as targets. Under assay conditions, this antibody wascytotoxic to all lymphocytes, and this cytotoxicity was partiallyinhibited with soluble HLA class I and HLA class II in a dose dependentmanner. (Table 2.1).

TABLE 2.1 HLA Class I Inhibition of Lymphocytotoxicity in AntiserumProduced to Purified HIV Lysates Anti-HIV & Anti-HIV & Anti-HIV &Anti-HIV & Pre- HLAI & II HLAI & II HLAI & II HLAI & II Immune DilutionAnti-HIV 25 ug 50 ug 100 ug 200 ug Serum u 8 8 8 8 8 0 1:5 8 8 8 8 8 01:10 8 8 4 4 4 0 1:20 8 8 4 4 4 0 1:40 8 8 4 4 4 0 1:80 8 4 2 4 2 01:160 8 4 0 2 0 0 1:320 8 2 0 0 0 0 1:640 8 0 0 0 0 0 1:1280 4 0 0 0 0 0

Soluble HLA class I&II was added to micro titer wells containing antiHIV antibody and incubated overnight at 4° C. Soluble HLA I&II reducedlymphocytotoxicity but had no additional effect at concentrations above50 ug.

Further studies demonstrated an antibody that reacted with aphylogenetically preserved carbohydrate antigen present on gp120, humanred and white blood cells, and red blood cells from different animalsincluding rabbit, rat, and guinea pig. Absorption studies with human andrabbit red cells completely removed the remaining antibody activity toboth RBC's (Table 2.2) and lymphocytes (Table 2.3) following absorptionwith S-HLA-I & II.

TABLE 2.2 Analysis of Anti RBC Absorbed Anti-HIV Antibody for RBCAgglutinins Cells RBC Absorp- Anti* Anti* Anti* tion HIV1 HIV2 HIV1 & 2# AB+ O+ rabbit AB+ O+ rabbit AB+ O+ rabbit 0 256 256 256 256 128 128128 256 128 1 128 128 128 128 64 128 64 128 128 2 32 32 32 32 32 64 3232 32 3 8 4 4 4 4 4 8 4 4 4 2 — — — — — 2 — — *Antisera produced topurified HIV lysate resulted in RBC agglutinins produced in goatsfollowing immunization. Goats (n = 2 each) were immunized with HIV1_(MN)and HIV1_(BAL) andor HIV2_(NZ), respectively.

TABLE 2.3 Analysis of Anti RBC Absorbed Anti-HIV Antibody forLymphocytotoxicity During Immunization RBC Absorp- Anti* Anti* Anti*tion HIV1 HIV2 HIV1 & 2 (n) # AB+ O+ rabbit AB+ O+ rabbit AB+ O+ rabbit0 512 512 256 1024 1024 1024 512 1024 1024 1 256 256 256 256 512 512 256256 256 2 128 64 128 64 64 128 64 128 64 3 32 16 16 16 16 32 16 16 16 44 4 4 4 4 4 4 4 4 5 2 2 2 ± 2 2 2 2 2 *Antisera produced to HIV withoutglycosidase treatment at 20 week blood collection date and absorbed 0-5times with red blood cells and tested against lymphocytes forlymphocytotoxicity.

Following the absorption and removal of antibody specificities to HLAand hemoagglutinins the goat anti-HIV antibody was identified to containspecificities similar to those previously described in the literature.This information demonstrated the need for modification of the HIVproteins to remove carbohydrate and HLA antigens to avoid the generationof cytotoxic antibodies directed against human antigens.

EXAMPLE 3 Purification and HLA Class I and Class II Antigen Removal andCarbohydrate Removal of HIV Protein Isolated from HIV Lysates

HIV lysates of HIV1_(MN), HIV1_(BAL) and HIV2_(NZ) were purchased fromAdvanced Biotechnologies, Columbia, Md. They contained proteinconcentrations in the range 0.8-1.2 mg protein/ml. Protease inhibitorswere added to protect the proteins from degradation, and non-ionicdetergent (Igepal Ca-630, or Nonidet P-40) was added to dissociate HIVproteins. The mixture was dispensed into capped extraction tubes with adelipidating reagent to remove lipids and clarify the mixture. Lipidswere differentially dissolved in the organic layer after centrifugationand the aqueous phase was removed.

Contaminants of cell culture (origin, including HLA, were removed byimmunoaffinity chromatography on five separate affinity matricesprepared by the covalent attachment of: immunoaffinity purifiedpolyclonal IgG antibodies to human serum proteins, monoclonal IgGantibody to HLA-1, monoclonal IgG antibody to HLA-2, monoclonal antibodyto β2-microglobulin, immunoaffinity purified polyclonal IgG antibodiesto lymphocyte and red blood cell membrane antigens. Each matrixcontained 2-3 mg antibody per g Sepharose CL6B. Columns were configuredin a tandem arrangement, and matrices were poured and equilibrated withPBS at pH 7.8 containing 0.1% Igepal Ca-630. Lysate solutions in volumeequivalent to column bed volume were successively chromatographedthrough each column at a flowrate of 10 ml/h. The chromatography andelution were monitored spectrophotometrically at a wavelength of 280 nm.The protein-rich non-binding fraction containing the HIV relatedproteins were concentrated to 1 mg protein/ml.

SDS was added to the immunoaffinity-purified mixture containing the HIVproteins of interest and the mixture was heated at 70° C. for 10minutes. The protein were enzymatically deglycosylated using PGNase. Theprotein mixture was fractionated by size chromatography on Sepharose G50pre-equilibrated with saline containing 0.1% non-ionic detergent.Protein fractions were collected and those containing the desired HIVproteins were individually pooled. The three protein pools, individuallyenriched for gp160 and gp120, p66/55 and gp41, and p24, p17 and p10 wereretained. Fractions were stored at −70° C. until further processing.

Detailed methods for quantitation and removal of human leukocyte antigen(HLA) are disclosed in “Identification, Characterisation, andQuantitation of Soluble HLA Antigens in Circulation and PeritonealDialysate of Renal Patients”, F. Gelder, Annals of Surgery Vol. 213,(1991), incorporated herein by reference.

Table 3.1 shows data obtained from the purification.

TABLE 3.1 Purification of HIV Lysate % Re- Fractionation Protein VolumeTotal HLA I HLA II cov- Step mg/ml ml Protein ng/ml ng/ml ery HIV Lysate1.13 10 11.3 1833 692 100% Post Lipid 0.96 10.3 9.8 1726 683 86.7Removal Post Affinity 0.44 19.5 8.58 <10 <10 75.9 Chromatography Post1.01 8.1 8.18 <10 <10 72.3 Concentration

HIV preparations purified as described typically were devoid ofcontaminants, including HLA class I or class II. SDS-PAGE with WesternBlot analysis of purified HIV preparations consistently demonstrated thepresence of gp160 , gp120, p66/55, gp41, p10, p24, p17 and p7 employingthe pooled human IgG anti HIV.

EXAMPLE 4

Preparation and Characterization of the Biochemical and ConformationalRequirements of MDP Microparticle for Eliciting Adjuvant Effects withPurified HIV

A multiple repeat of muramyl dipeptide (MDP) isolated fromPropionibacterium acini, formed the core structure of the MDPmicrocarrier complex of this example. The chemical composition of themonomeric subunit is

MDP has well known immunostimulatory properties which have beenextensively evaluated in studies designed to determine its effect onincreasing immune function. Those skilled in the art are familiar withthis effect.

To date, both MDP isolated from natural sources and synthetic MDP havebeen associated with significant toxicity when administered to mammals.This toxicity has limited the effectiveness of MDP as a carrier.

A method for the isolation of MDP free from toxic components is providedherein. Propionibacterium acini was grown to a mid-stationary growthphase and washed to remove contaminants of bacterial culture originemploying techniques well known to those in the art. Hydrophobiccomponents contained in the cell walls and cytoplasm were sequentiallyextracted by successive washes in gradual concentrations ofethanol/methanol/water at elevated temperatures. The resulting MDPmicroparticle was suspended in 10% ethanol and its concentration wasmeasured by relating its absorbance at 540 nm to the absorbance ofturbidity standards. The concentration of the MDP microparticle wasadjusted to 1 mg/ml for storage and later use.

Analysis of this preparation demonstrated muramyl dipeptide extensivelycrosslinked with a microparticle size of 0.1 to 0.2 micron. The terminaldipeptide amino-linked L-alanine-D-isoglutamine was identical to themonomeric structure shown above. It is well known that there can bedifferences between bacterial strains and these differences can resultin differences in peptide composition, such as terminal peptides withfive or more amino acids, changes in dipeptide amino acid composition,in particular L-alanine-L-isoglutamine, and sites where O-acylated betamyristate groups have been incorporated. These are not desirable andaccount for toxicity and poor adjuvant properties of MDP isolated fromnatural sources. In a preferred embodiment, the MDP microparticles(0.01-0.2 micron; preferrably 0.05-0.1) have amino-linkedL-alanine-D-isoglutamine dipeptide. Such a microparticle can be isolatedfrom natural sources, as above, or synthesized using well knownsynthetic procedures.

EXAMPLE 5 Preparation and Preliminary Evaluation of MDP-ImmunogenConjugate

The adjuvant effect of the MDP microparticle (0.2 μ) of the precedingexample or antibody production was evaluated employing a poorlyimmunogenic monoclonal human lambda light chain fragment lackingapproximately 22 amino acids at the sulfhydryl bridge (Mr˜18000) as theimmunogen (I). Two conjugates were made, one in which the immunogen wascovalently conjugated to MDP through the carboxyl terminal group and onewherein conjugation was through the amino terminal group. MDP-immunogenconjugates were assembled in a stepwise manner and reagent exchange wasperformed after each step by centrifugation, supernatant removal andreplacement with the required reagent to continue the conjugationsequentially through MDP:immunogen assembly. The molar ratios that areshown with each reaction and the reagent exchange that was performedafter each step prevented multiple point attachment of the immunogenwhich, from preliminary experiments, significantly reduced immuneantibody responses.

Synthesis of MDP:NH₂:Immunogen:CO₂H

Protocol for Efficient Two-Step Coupling of HIV Proteins to MuramylDipeptide Using EDC

The following procedure, adapted from a procedure described by Grabarek,Z. and Gergely, J., J. Anal. Bicohem. 185:1311 (1990), allows forsequential coupling of HIV proteins and peptides to MDP without exposingthe HIV protein to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)and thus affecting carboxyls on HIV. This procedure calls for quenchingthe first reaction with a thiol compound. The reaction is carried out in2-[N-morpholino]ethane sulfonic acid (MES) (pH 4.5-5.0).

MDP (10 mg) lyophilized from water resuspended in MES (0.5 ml) (pH4.5-5.0) and 0.5ml EDC (0.5 mg˜2 mM) dissolved in MES (pH 4.5-5.0) werecombined and reacted for 15 minutes at room temperature.2-mercaptoethanol (final concentration of 20 mM) was added to quench theEDC and separated by centrifugation. The reaction mixture was washedonce with MES and resuspended in 0.5 ml MES (pH 4.5-5.0). The human λlight chain fragment dissolved in MES was added to the activated MDP ata molar ratio of about 2:1. The PH of the reaction was slowly increasedover a 15 minute period to 8.5 by the addition of MES (0.5M pH 8.5) andreacted for 2 hours at room temperature. The concentration of the λlight chain fragment added to MDP was calculated from quantitativeanalysis of MDP terminal CO₂H group and expressed as mole CO₂H per mgMDP. The reaction was quenched by adding hydroxylamine to a finalconcentration of 10 mM. This method of quenching hydrolyzed anyunreacted MDP activation sites and resulted in regeneration of theoriginal carboxyls. Other means of quenching involve adding 20-50 mMTris, lysine, glycine, or ethanolamine; however, these primaryamine-containing compounds will result in modified carboxyls MDP. Whenthe MDP is used with an HIV synthetic peptide and modification of thatpeptide is desired, such as to change hydrophobicity or to attachbio-active compounds, the modification can be attained by adding thedesired compound prior to the final quench step. This permits thedesired compounds to be added sequentially following the initialcoupling with HIV peptides. Bioactive peptides also could be added atdesired ratios with HIV peptides as a single step when greater controlof peptide immunogeneicity is desired. Biological response modifierssuch as IL2 are well known to those skilled in the art and could be usedfor this purpose.

Separation was achieved by centrifugation, a wash step, and resuspensionin the buffer of choice.

PREPARATION OF MDP—CO₂H-IMMUNOGEN—NH₂

Synthesis of MDP:NH₂CH₂CH₂NH₂:CO₂H:Immunogen:NH₂

Protocol for Efficient Three-Step Coupling of Proteins to MuramylDipeptide Using EDC

This procedure allows for sequential coupling of a protein or peptidesto MDP without exposing the protein to EDC and thus affecting aminogroups on the protein. The procedure employs two intermediate stepsconducted sequentially. The initial reaction is carried out in MES (pH4.5-5.0). MDP (10 mg) lyophilized from water resuspended in 0.5 ml MESpH4.5 and 0.5 ml EDC (0.5 mg˜2 mM) dissolved in MES were combined andreacted for 15 minutes at room temperature. Excess EDC was quenched bythe addition of 2-mercaptoethanol (final concentration of 20 mM), andthe activated MDP was separated by centrifugation, washed two times withMES and resuspended in 0.5 ml MES (pH 4.5). Diaminoethane(NH₂CH₂CH₂NH₂), dissolved in MES(pH4.5) was added to the activated MDPat a molar ratio of about 10:1. The pH was slowly increased over a 15minute period by the addition of MES 0.5M pH8.5 and reacted for 1 hourat room temperature. MDP:NH₂CH₂CH₂NH₂ was separated by centrifugation,washed two times with MES and resuspended in 0.5 ml MES pH 4.5. The λlight chain fragment was suspended in 0.5 ml MES pH4.5, and 0.5 ml EDC(0.5 mg˜2 mM) dissolved in MES was added and reacted for 15 minutes atroom temperature. Excess EDC was quenched by the addition of2-mercaptoethanol (final concentration of 20 mM) and the activatedprotein was separated from excess reducing agents and inactivatedcrosslinkers by size chromatography on an appropriate size gelfiltration column. The activated protein was added to the activatedMDP:NH₂CH₂CH₂NH₂ at a molar ratio of about 5:1 and reacted for 2 hoursat room temperature. The concentration of protein added to MDP wascalculated from quantitative analysis of MDP terminal CO₂H group andexpressed as mole CO₂H per mg MDP. The reaction was quenched by addinghydroxylamine to a final concentration of 10 mM. This method ofquenching hydrolyzed any unreacted MDP activation sites and resulted inregeneration of the original carboxyls. If an HIV synthetic peptide isused as the immunogen and modification of that peptide is desired, suchas to change hydrophobicity of that peptide or attach bioactivecompounds, the modification can be accomplished as described above.Separation was achieved by centrifugation, a wash step, and resuspensionin the buffer of choice.

It should be noted that bioactive compounds may require the intermediatestep described above when attachment through the CO₂H group is desired.

EXAMPLE 6 Comparative Study of MDP Immunogen Conjugate AgainstCommercial Adjuvants Including Freunds Complete Adjuvant, RIBI®, TiterMax® and Alum

The adjuvant effect of this MDP microparticle (0.1μ) on antibodyproduction was evaluated employing the poorly immunogenic monoclonalhuman lambda light chain fragment described in the preceding example(Mr˜18,000) as the immunogen (I). Two conjugates were prepared, one inwhich the immunogen was covalently conjugated to MDP through thecarboxyl terminal group and one whereon the conjugation was through theamino terminal group.

Rabbits (n=5 each group) were immunized subcutaneously withapproximately 100 micrograms of lambda light chain attached to 500microgram MDP and emulsified in squalene. Animals were immunized atmonthly intervals and test bleeds were obtained prior to immunizationand at two week intervals throughout. The antibody responses toMDP:NH₂—I—CO₂H and MDP:NH₂CH₂CH₂NH₂:HO₂C—I—NH₂ were comparable inactivity; however, the MDP:NH2—I—CO₂H—stimulated rabbits produced atleast one additional antibody specificity determined by competitive EIA.The antibody responses obtained were compared to those obtained whenconventional adjuvants were employed for antibody response, includingFreund's complete adjuvant, Ribi®, Titer Max® and Alum (aluminumhydroxide). Both MDP:HO₂C—I—NH₂ and MDP: NH₂—I—CO₂H were significantlysuperior to the conventional adjuvants with immunogen in inducingantibody. Immunogen concentration (100 μg/immunization) and immunizationschedule were identical in all groups. Table 6.1 shows the antibodytiter measured at bi-weekly intervals and titer is expressed as thereciprocal of the dilution producing a positive reaction as describedabove. Both MDP conjugates were superior to conventional and well knownadjuvants.

TABLE 6.1 *MDP:I +MDP:I Freund's Titer Week CO₂H NH₂ Complete Ribi MaxAlum T-C — — — — — — 2 — — — — — — 4 2 — — — — — 6 16 4 — — — — 8 256128 — — — — 10 512 512 4 4 4 — 12 1024 512 16 16 16 — 14 4096 2048 32 3264 4 16 409E 4096 64 128 256 8 18 8192 4096 256 512 1024 32 20 163848192 256 512 2048 32

T-0=Primary immunization and pre-immunization blood collection.

MDP:I muramyl dipeptide:immunogen micro particle (≦0.2μ).

* Peptide was conjugated at amino terminal group to isoglutamine withcarboxy terminus exposed. (MDP:I:CO₂H)

+ Peptide was conjugated at carboxy terminus through an intermediatestep employing diaminathene to modify the carboxyl terminus of MDP.(MDP:I:NH₂)

EXAMPLE 7 Cytokine Response of Peripheral Blood Mononuclear CellsInduced with MDP:NH₂:I:CO₂H and MDP:NH₂CH₂CH₂NH₂: O₂HC—I—NH₂Immunogens

To further evaluate the mechanisms associated with the increasedantibody response to the MDP microparticle-immunogen complexes, an invitro method which measured cytokine production of peripheral bloodmononuclear cells was employed and compared to known cytokine inducers.Lipopolysaccharide (LPS) and LPS with phytohemagglutinin (PHA) wereemployed as known cytokine inducers. Cytokines were quantitatedemploying a well-established assay and expressed as units/ml. Peripheralblood mononuclear cells were isolated by Ficol Hypaque gradientcentrifugation and adjusted to a concentration of 2×10⁶/ml in tissueculture media. Cells (100 μl) were plated into microculture wells.MDP:I:CO₂H, MDP:I:NH₂, PHA+LPS, LPS and media alone were added undilutedor diluted 1:10 and 1:25 (10 ul). Cultures were incubated at 37° C. in a5% CO₂ atmosphere for 48 hours and supernatants were removed and assayedby standard bioassays and/or EIA methods.

TABLE 7.1 IFNR IL2 TNF IL6 MDP:I:CO₂H 550 510 152 62 MDP:I:NH₂ 520 495176 73 LPS + PHA 340 320 495 450 LPS 210 150 325 310 MEDIA 0 0 0 0I:CO₂H 496 398 68 25 NH₂ 23 410 72 21 LPS + PHA 205 165 210 152 LPS 17590 135 140 I:CO₂H 125 90 5 10 I:NH₂ 51 85 9 10 LPS + PHA 20 10 15 20 LPS20 10 15 20

Both MDP:I:CO₂H and MDP:I:NH₂ stimulated greater Type I cytokineresponses than LPS+PHA or LPS alone. Type 1 cytokine responses enhancedimmune events while Type 2 cytokine response is indicated by elevatedIFN gamma and IL2 and lower levels of TNF and IL6.

EXAMPLE 8 Characterization of the Antibody Response in Goats to HIVProteins Untreated and Treated to Remove Carbohydrates Moieties andComparison of the Adjuvant Properties of MDP Microparticles withConventional Adjuvants Example 8.1

HIV1_(MN), HIV1_(BAL) and HIV2_(NZ) viral lysates were purchased fromAdvanced Biotechnologies Inc., Columbia, Md. and one-half of eachpreparation was treated enzymatically to remove carbohydrate and allpreparations (with and without carbohydrate) were purified as above. Analiquot of each was conjugated to MDP through the amino terminal residuein accordance with the procedures set forth in Example 5, andindividually suspended in squalene. For comparison, these HIV proteinsalso were emulsified in Freund's complete adjuvant without conjugationto MDP.

Group 1—HIV1_(MN): HIV1_(BAL), 1:1, without carbohydrate removal;

Group 2—HIV1_(MN): HIV1_(BAL), 1:1, with carbohydrate removal;

Group 3—HIV2_(NZ) without carbohydrate removal;

Group 4—HIV2_(Nz), with carbohydrate removal

Group 5—HIV1_(MN): HIV1_(BAL): HIV2_(NZ), 1:1:1, without carbohydrateremoval;

Group 6—HIV1_(MN): HIV1_(BAL): HIV2_(NZ), 1:1:1, with carbohydrateremoval;

Group 7—HIV1_(MN): HIV1_(BAL), HIV2_(NZ), 1:1:1, without carbohydrateremoval and emulsified in Freund's complete adjuvant without conjugationto MDP.

Group 8—HIV1_(MN): HIV1_(BAL): HIV2_(NZ), 1:1:1, with carbohydrateremoval and emulsified in Freund's complete adjuvant with conjugation toMDP.

Goats were stratified into immunization groups 1-8 (n=3 each) andrespectively immunized at the intervals shown in Table 8.1 with 100 μgHIV/immunization. Blood samples were obtained prior to immunization andat biweekly intervals. Antibody reactivity was quantitated by EIA usingthe FDA approved commercially available test kit from AbbottLaboratories. Results were expressed as the reciprocal of the antiseradilution that produce an absorbance value >1.0.

TABLE 8.1 Analysis of Anti HIV Antibody Response to HIV Proteins WeekGroup 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 # AntiHIV1 Anti HIV1¹ Anti HIV2 Anti HIV2² Anti HIV1 & 2 Anti HIV1 & 2¹ AntiHIV1 & 2 Anti HIV1 & 2 T-O* 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 4* 8 8 8 48 8 0 0 6 64 64 32 16 64 128 2 0 8* 64 128 32 32 64 128 4 2 10 512 51264 128 256 512 16 8 12* 512 1024 128 256 512 1024 16 16 14 2048 2048 512512 1024 2048 32 32 16* 4096 4096 256 512 4096 4096 64 32 18 4096 8192512 1024 8192 8192 128 54 20 8192 16384 1024 1024 16384 16384 128 64 T-O= Primary immunization and pre-immunization blood collection¹carbohydrates removed *Immunization & Booster

The antibody responses were measured at bi-weekly intervals throughoutimmunization. There was no significant difference in antibodyreactivity, measured by EIA, between individual animal groups (Groups1&2, 3&4, 5&6) with MDP. Carbohydrate removal had no effect onproduction of antibody to the desired anti-HIV proteins.

Example 8.2

Antisera obtained as described in Example 8.1 were evaluated forhemagglutinating antibodies. As can be seen from Table 8.2, carbohydrateremoval from the HIV proteins obliterated the hemagglutination response.

TABLE 8.2 Analysis of Anti HIV Antibody Response to Red Blood Cells WeekGroup 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 # AntiHIV1 Anti HIV1¹ Anti HIV2 Anti HIV2² Anti HIV1 & 2 Anti RIV1 & 2¹ AntiHIV1 & 2 Anti HIV1 & 2¹ T-O* — — — — — — — — 2 — — — — — — — — 4* — — —— — — — — 6 2 — 2 — 4 — 4 — 8* 4 — 4 — 4 — 8 — 10 16 — 8 — 8 — 16 — 12*32 — 8 — 32 — 16 — 14 64 — 64 — 128 — 64 — 16* 64 — 64 — 64 — 128 — 18128 — 128 — 128 — 256 — 20 256 — 256 — 128 — 256 — T-O = Primaryimmunization and pre-immunization blood collection ^(l)Carbohydratesremoved *Immunization & Booster

HIV preparations treated to remove carbohydrate groups failed to produceantibody reactivities able to agglutinate red blood cells (Table 8.2A).Goats immunized with MDP-HIV conjugates without carbohydrate depletionand a goat immunized with purified HIV without carbohydrate depletionemulsified in Freund's complete adjuvant produced red cell agglutinins.There was no detectable difference in the titer of red cell agglutininsin antisera from goats immunized with MDP-HIV conjugates or HIVemulsified in Freund's complete adjuvant. The red cell agglutininsdescribed herein were essentially identical to those described in thepreliminary studies of Example 2. These agglutinins were cytotoxic(Table 8.2A). However, there was no detectable antibody reactivity toHLA class I or class II and absorption with red blood cells completelyremoved both the hemagglutinating and the cytotoxic antibody reactivity.

TABLE 8.2A Sequential Analysis of Anti HIV Antibodies forLymphocytotoxicity Week Group 1 Group 2 Group 3 Group 4 Group 5 Group 6Group 7 Group 8 # Anti HIV1 Anti HIV1¹ Anti HIV2 Anti HIV2² Anti HIV1 &2 Anti HIV1 & 2¹ Anti HIV1 & 2 Anti HIV1 & 2¹ T-O* ± ± ± ± ± ± ± — 2 ± ±± ± ± ± ± — 4* +4 ± +2 ± +4 ± ± — 6 +8 ± +2 ± +8 ± +2 — 8* +8 ± +4 ± +16± +4 — 10 +64 ± +32 ± +32 ± +4 — 12* +256 ± +256 ± +128 ± +16 — 14 +256± +512 ± +256 ± +32 — 16* +512 ± +512 ± +512 ± +64 — 18 +512 ± +1024 ±+512 ± +128 — 20 +512 ± +1024 ± +1024 ± +128 — T-O = Primaryimmunization and pre-immunization blood collection ¹Carbohydratesremoved *Immunization & Booster

To evaluate the possibility of mimicry between HIV and RBC carbohydrategroups, additional antisera were produced by immunizing a goat with animmunogen composed of purified and pooled cell membranes isolated fromhuman red blood cells. Western Blot analysis employing this antiserademonstrated reactivity with a red blood cell glycoprotein (˜Mr35,000)and reacted with HIV gp41 and gp120 . However, HIV gp41 and gp120treated to remove carbohydrate groups were unreactive. These data wereconsistent with phylogenetic mimicry between carbohydrate epitope on HIVand red blood cell glycoproteins.

Western Blot analysis demonstrated strong reactivity to most HIVproteins, including gp160, gp120, gp41, p66/55, p10, p24, p17 and p7.There was no apparent difference in the reactivity or specificity ofthese antibodies to the HIV epitopes disclosed herein. These antibodiesreacted to all HIV isolates tested, including those which have beenshown to have resistance to reverse transcriptase and proteaseinhibitors.

Example 8.3

Neutralization of HIV Infectivity by Antibodies produced toCarbohydrate-Depleted HIV

This example describes and characterizes the neutralization of HIVinfectivity using the antibodies produced to carbohydrate depleted HIVdisclosed above. The results indicate that these antibodies contain highlevels of neutralizing activity and protect CEM cells from infection ina dose dependent manner. Neutralization Assay:

A sensitive neutralization assay was employed to quantitate the effectof goat anti HIV on HIV infectivity. The CEM CD4⁺ cell line, which ishighly susceptible to HIV infection, was chosen as the target cell todetermine the effect of this antibody on HIV infectivity. The antibodyand dilutions were made as required in RPMI medium containing 10% fetalcalf serum. A suspension of HIV1_(SF2) was harvested from about four-daycultures of CEM in log growth phase, filtered through 0.2 or 0.45 micronfilters, aliquoted, and frozen at −70° C. One aliquot was thawed,titrated to determine the TCID₅₀, and subsequent assays were performedwith freshly thawed aliquots, diluted 1:500 in culture medium to aconcentration of approximately ten times the amount required to infect50% of CEM cells in culture (10 TCID₅₀). The virus suspension was mixedwith an equal volume (250 ul) of five-fold dilutions of antibody from1:5 to 1:9,765,625. The virus/antibody mixture was incubated for 60minutes at 37° C. and duplicate samples of 200 ul used to inoculatewells containing 1.0 ml of approximately 2×10⁵ CEM cells per well. Thecultures were incubated at 37° C. in a humidified, 5% CO₂ atmosphere for14 days. The cells were harvested, pelleted, and lysed with 1% TritonX-100 in PBS for about 10 minutes. The amount of virus (or viralantigen) present in lysed cells was quantitated using a commerciallyavailable p24 assay. The titer of neutralizing activity was determinedas the reciprocal of the dilution of antibody which inhibited p24antigen production by greater than 50% of virus control culturesincubated without antibody, or with goat pre-immune IgG prepared in asimilar manner. Two hundred microliters of the lysed cellular suspensionwere assayed.

TABLE 8.3A Analysis of Anti HIV Antibody Neutralization Activity WeekGroup 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Group 8 # AntiHIV1 Anti HIV1¹ Anti HIV2 Anti HIV2² Anti HIV 1 & 2 Anti HIV 1 & 2¹ AntiHIV 1 & 2 Anti HIV 1 & 2 T-O* — — — — — — — 2 — — — — — — —  4* — — — —— 4 — — 6 32 16 — — 16 16 — —  8* 256 32 4 — 64 128 2 — 10  1024 128 4 4512 256 8 — 12* 1024 512 16 8 2048 1024 32 — 14  5096 1024 256 32 50962048 64 — 16* 10192 2048 512 64 10192 2048 128 — 18  20384 2048 1024 12820384 5096 128 —  20** 20384/204 5096/5096 2048/256 256/256 40766/1019210192/10192 256/12 — T-O = Primary immunization and pre-immunizationblood collection ¹Carbohydrates removed *Immunization & Booster ** =anti-HIv preparations obtained from the week 20 bleeds were absorbedwith human red blood cells and both unabsorbed and absorbed samples weretested under identical conditions. Results are expressed as# (unabsorbed/absorbed) neutralization titer.

The anti HIV preparations with the greatest neutralizing activity wereproduced to HIV-MDP conjugates that were not treated to removecarbohydrate determinants (Table 8.3A). Red blood cell absorption ofantibodies to carbohydrate-depleted HIV conjugates had no effect.However, RBC absorption of antibodies to carbohydrate-intact HIVconjugates resulted in a significant reduction in neutralizingreactivity, confirming the presence of phylogenetically presentcarbohydrate moieties shared between HIV and humans (bottom row, Table8.3A). All anti-HIV antibody preparations produced to HIV-MDP conjugateswere statistically greater than anti-HIV produced using Freund'scomplete adjuvant. Due to the predicted genetic variabilitycharacteristic of HIV, anti-HIV from the 20 week preparations weretested for neutralizing activity using HIVMN and four HIV wild isolates,including one characterized as a multi-drug resistant strain underconditions identical to those described above. Anti-HTV from the 20 weekantibody preparations produced to HIV conjugates devoid of carbohydrateneutralized all strains (Table 8.3B).

Those skilled in the art will recognize that the neutralizing activityproduced to HLA-depleted, carbohydrate-depleted HIV proteins isconsiderably higher than the neutralizing activity typically observed inhuman anti-HIV sera.

TABLE 8.3B Analysis of Anti HIV Antibody Neutralization Activity AgainstMultiple Strains HIV Strain/ Anti Anti Anti Pre Immune Isolate HIV1¹HIV2¹ HIV1&2¹ IgG HIVSF 10192 512 20384 0 HIVMN 5096 256 10192 0 Wild#110192 512 2C384 0 Wild#2 2048 256 5096 0 Wild#3 2048 128 5096 0 1 =carbohydrates removed

Example 8.4

Effect of Anti HIV on HIV Infected CD4 Lymphocytes in an AntibodyDependent Complement Mediated Cytotoxicity Assay

Antibodies against carbohydrate-depleted and carbohydrate-intact HIVconjugates were evaluated for complement mediated cytotoxicityreactivity to normal peripheral blood mononuclear cells enriched for CD4lymphocytes with and without infection with HIV. Normal peripheralmononuclear cells were isolated, subjected to Ficol Hypaque gradientcentrifugation and enriched for CD4⁺ lymphocytes. CD4⁺ lymphocytes werestimulated with PHA and infected with HIV_(MN) for 7 days in microculture. Supernatants were removed and replaced with anti HIV producedto HIV preparations following sugar group removal and shown to have nocytotoxic effects on normal cells. As can be seen in Table 8.4, anti HIVlysed infected CD4 lymphocytes in a dose dependent fashion.

TABLE 8.4 Analysis of Anti HIV Antibody Mediated Cytotoxicity ofInfected CD4 Lymphocytes Week Group 1 Group 2 Group 3 Group 4 Group 5Group 6 Group 7 Group 8 # Anti HIV1 Anti HIV1¹ Anti HIV2 Anti HIV2² AntiHIV1 & 2 Anti HIV1 & 2¹ Anti HIV1 & 2 Anti HIV1 & 2¹ T-O* ± ± ± ± ± ± ±— 2 ± ± ± ± ± ± ± —  4* 4 4 2 ± 4 4 ± — 6 8 16 2 ± 32 32 ± —  8* 32 32 44 256 256 2 — 10  128 64 32 32 512 512 8 — 12* 256 128 256 64 1024 102416 — 14  1024 512 512 256 2048 2048 16 2 16* 2048 1024 512 256 4096 409632 4 18  4096 2048 1024 512 8192 8192 64 8 20  8192 4096 1024 512 81928192 256 8 T-O = Primary immunization and pre-immunization bloodcollection ¹Carbohydrates removed *Immunization & Booster

Example 8.5

Synthesis of Peptides Corresponding to the Amino Acid Sequence of HIVProteins

Synthetic peptides were constructed as twelve-mer peptides which mimicthe amino acid sequence of HIV1_(SF2). Amino acid sequences for gp 120,gp 41, Vif, gag p 17, gag p 24, nef, Rev, Integrase, Protease, Tat:HxB2and Reverse Transcriptase and Reverse Transcriptase with overlaps by sixamino acid residues were synthesized by and purchased from PurificationSystems, Inc. employing solid phase technology.

Butyloxycarbonyl-S-4-methylbenzyl-L-cystine coupled to polystyrene usingdicyclohexylcarbodiimide with a catalytic amount of4-N,N-dimethylaminopyridine was used as the solid-phase support for thesynthesis. The amino groups were protected with tert-butyloxycarbonyl(t-BOC) and the side chain protecting groups were as follows: benzylether for the hydroxyl of serine, dichlorobenzyl ether for the phenolichydroxyl of tyrosine, and the beta benzyl-esters were used for thecarboxyl groups on glutamic acid and aspartic acid, respectively.Trifluoroacetic acid (40% in CH₂Cl₂) was used to remove t-BOC and theresulting salt was neutralized with N-diisopropylethylamine (10% inCH₂Cl₂)

Diisopropylcarbodiimide was used to couple the t-BOC amino acids. Theprotecting groups were removed and the peptide was cleaved from theresin at 0° degrees C. with anhydrous hydrogen fluoride containing 10%an sole and 1% ethanedithiol as scavengers. The hydrogen fluoridereagent was removed under vacuum at 0° C. and the peptide then wasprecipitated and washed with anhydrous ether. After extraction of thepeptide from the resin with trifluoroacetic acid, the solvent wasevaporated to 15° C. and the peptide was again precipitated with ether.The ether was decanted after centrifugation and the pellet was dissolvedin 5% acetic acid with 6 M guanidine HCl. This solution was desalted ona BioGel P2 column equilibrated in 5% acetic acid and the peptidecontaining fractions were pooled and lyophilized. A cysteine residue wasadded to the carboxyl terminus of the peptide as needed to provide afunctional SH group for the coupling of the peptide to carrier proteinsor to a solid support for EIA procedures or to MDP (Example 5). Whenmultiple repeats of the peptide were desired, synthesis was conducted byfirst attaching a cysteine residue to the resin support. Carbon spacersof various lengths were added; the choice of spacer length varied andwas dependent on the application, peptide charge and length and stericinfluences predicted from preliminary data resulting from peptideattachments to supports. A six carbon spacer such as 6-aminohexanoicacid was first attached with lysine- (lysine)2-(lysine)4 additions asdescribed above with diaminoethane in Example 5 but altering thesequence of protective group blocking. Amino groups were protected andthen deprotected to permit two lysine residues to attach to thedeprotected amino terminus, deprotection followed by lysine additionbuilt a branched chain structure for peptide synthesis. Peptides withspecific biological function or with sequences that are susceptible toenzymatic degradation were modified by the addition of D-amino acids.One particularly useful addition is the addition ofL-alanine-D-isoglutamine with the peptide of interest synthesized off ofthe NH₂ terminus of D-Isoglutamine. In another arrangement, the peptidewas synthesized with L-Lysine-L-Lysine-peptide-D-Isoglutamine. Thecarboxy terminal lysine groups are highly susceptible to enzymedegradation by many enzymes in the micro environment whileD-isoglutamine both results in an increase in half life of the peptideand provides a hydrophobic site to assemble peptides that requireamphipathic properties to elicit a function such as receptor binding andimmune induction through MHC associated events. A tyrosine residue wasadded to the amino terminus for radioactive labeling with ¹²⁵Iodine todetermine peptide-to-carrier protein coupling efficiency and to identifythe peptide during purification. ¹²⁵I also provided a tracer to followthe half life of the peptide in biological systems and evaluate receptorbinding when peptide function was not affected by tyrosine addition.

Example 8.6

The Use of Synthetic Peptides which Mimic Peptide Sequences of HIV andOther Retroviruses, for the Identification of Viral Epitopes

Within this invention, synthetic peptide sequences which mimic highlyconserved sequences found in HIV and other retroviruses are disclosedand their functional significance as immunological targets in treatingviral infections are identified. These peptides have further applicationin the diagnosis and management of HIV infection resulting from HIVmicrovariants with sequences that contribute to the pathogenesis of HIVthrough nonspecific down regulation of immune reactions, induction ofautoimmunity, and through toxic effects leading to HIV-associatedperipheral neuropathy. Each of these events, when presented in apatient, contribute to the pathogenesis of HIV and decline in thequality of life. Synthetic peptides employed to identify and quantitatethose HIV-peptide regions which initate those events, have utility inidentifying risk factors for autoimmunity and peripheral neuropathy. Thesynthetic peptides provide further utility in a novel assay procedure tomonitor disease progression and changes in progression as a result oftreatment.

In one step of this invention, an enzyme immunoassay (EIA) wasconfigured for the identification of goat antibody specificities on HIVnot recognized by human anti-HIV. Purified preparations of HIV1 gp120and gp41 proteins were coated on wells of polyvinyl microtitre plates at5 ug/ml in phosphate buffered saline (PBS, pH 7.8) by incubation fortwenty hours at 30° C. The wells were washed with PBS containing 0.1%Tween 20 (PBS/Tween) and the unoccupied sites of each well weresaturated with 5% bovine serum albumin by incubation for 1 hour at 37°C. The plates were used Immediately or stored at 40° C. Human IgGanti-HIV (100 ul) (Example 1) was added to each well, incubated for 25hours at 4° C., and the wells were washed with PBS/Tween. Goat anti-HIVpreviously produced and labeled with HRP by standard procedures wasadded to the wells at a dilution required to yield an absorbance of 1.0in the conditions of the assay (1:10000 titre) and incubated for 24hours at 40° C. The wells were washed and substrate added to determineamount of binding of the goat IgG. The percent inhibition of bindinginduced by blocking HIV proteins with human anti-HIV was calculatedusing the formula. (OD not blocked-OD blocked) OD not blocked×100−ODblocked negative control

Minimal blocking of goat anti-HIV-HRP conjugate by human anti-HIV wasindicative of binding of goat anti-HIV different from human anti-HIV.

In another step, an EIA was configured to identify epitopes on HIVproteins that were antigenic with goat antibodies but not with humanantibodies. Purified preparations of HIV1 gp120 and gp41 proteins werecoated on polystyrene beads at 5 ug/ml in PBS by incubation for twentyhours at 37° C. Beads were washed with PBS containing 0.1% Tween 20(PBS/Tween) and the unoccupied sites on each bead were saturated with 5%bovine serum albumin by incubation for 1 hour at 37° C. Beads were usedimmediately or stored at 4° C. Human IgG anti-HIV (100 ul) (Example 1)was added to each bead, incubated for 24 hours at 4° C. Human IgGanti-HIV (100 ul) (Example 1) was added to each bead, incubated for 24hours at 4° C. and the beads were washed with PBS/Tween. Syntheticpeptides were dissolved in PBS containing bovine serum albumin (5 mg/ml)and Tween 20 (0.1%) at a concentration of 0.1 mg/ml. The peptides (5 ul)were added to goat anti-HIV IgG-HRP conjugate solution (100 ul) andincubated for 24 hours at 40° C. The mixture then was added to two setsof beads coated with HIV. One set was blocked with human anti-HIV thatwas added to two sets of beads coated with HIV. One set was blocked withhuman anti-HIV that was added at the same time as the peptides wereadded to the goat anti-HIV-HRP conjugate. The beads with reactants wereincubated for 24 hours at 4° C. Following incubation, the beads werewashed and peroxidase activity was measured as described above. Activitywas plotted against peptide position within the HIV proteins. Theseplots showed areas of the HIV proteins targeted by goat HIV immune IgGthat was not recognized by human antibody. When inhibition of bindingwas observed with a specific synthetic peptide, additional peptides weresynthesized to overlap the original peptide by peptides of additionallengths. The lack of inhibition by the synthetic peptides was consideredto represent lack of immunologic targeting by the goat immune system.Employing this procedure, linear peptide epitopes were selected forstudy.

In another preferred procedure of this invention, an EIA was configuredto utilize peptide-peroxidase conjugates, produced as disclosed below,to identify antibody reactive epitopes on HIV proteins recognized by thegoat but not by the human immune system. Peptides mimicking HIVsequences were covalently attached to HRP to produce an enzyme labeledpeptide library for use in mapping antibody specificity. Specifically,HRP was dispensed into a reaction tube at a calculated molar ratio of 1part HRP:10 parts peptide. Each peptide to be coupled was individuallydispensed into microtubes at a concentration of 0.1-1 umole/ml atvolumes of 0.1-1 ml, each of which varied dependent upon quantity ofconjugate desired and molecular weight of peptide. HRP at aconcentration of 1-10 umole/ml was dissolved in 0.1 Mcarbonate/bicarbonate buffer (pH 9.8) at 4° C. and sodium periodate wasadded to achieve a final concentration of 0.02 M. The mixture wasimmediately dispensed into the microtubes containing the peptides mixedand reacted for 30 minutes. Ethylene glycol (0.02 M) was added to quenchremaining sodium periodate. The intermediate Schiff's base formedbetween the amino terminus of the peptide and the aldehyde formed byoxidation of the HRP carbohydrate moieties was reduced by the additionof sodium borohydride (0.2 M) in water. Chromatography of eachpeptide-peroxidase conjugate in Sephadex G25 was used to removereactants and excess peptide.

In this preferred assay, the antibody was used to bridge epitopesidentified on the HIV proteins prepared from HIV virus lysates andsynthetic HIV peptides covalently attached to HRP since antibodyreactive with HIV epitopes retained an antigen-binding site which couldreact with synthetic peptide epitopes attached to peroxidase. Beads werecoated with HIV proteins as described above. Coated beads were reactedwith goat anti-HIV IgG for 24 hours, the beads were washed and reactedwith substrate to determine peroxidase activity and, therefore, peptidebinding. With this procedure, only exact epitopes contained within thesynthetic peptide were recognized. In this assay, the completerepertoire of goat rectivities was identified. Human anti-HIV and goatanti-HIV reactivities were compared and peptides that reacted only withgoat anti-HIV were selected as candidate epitope targets. These datawere not considered to be all inclusive of differences between human andgoat reactivity to HIV proteins since differences between reactivitiesand shifts between the amino acid peptide sequence homologies on the HIVprotein could result in missing epitope reactivity secondary to theselection of the synthetic peptide.

EXAMPLE 9 Antibody Efficacy as a Therapeutic Agent

A study was conducted with a group of immunochemically designedantibodies to determine the efficacy of the antibodies as a therapeuticagent for the treatment of HIV/AIDS. Specifically, a mixture of lysatesof HIV isolates HIV1_(MN), HIV1_(BAL) and HIV2_(NZ), was treated as inExample 3 to remove low molecular weight contaminants and HLA class Iand class II antigens and to deglycosylate the HIV proteins. The proteinmixture was assayed and found to comprise peptides which mimic thefollowing regions of HIV1_(SF2) proteins:

gp120: an epitope region extending from amino acid residue 4-27 and asecond epitope region extending from amino acid residue 54-76;

gp41: an epitope region extending from amino acid residue 502-531;

reverse transcriptase heterodimer p66/55: an epitope region extendingfrom amino acid residue 254-295;

protease p10: an epitope region extending from amino acid region 69-94;

Gag gene protein p24: an epitope region extending from amino acid region166-181;

Gag gene protein p17: an epitope region extending from amino acid region2-23 and a second epitope region extending from amino acid region89-122; and

Gage gene protein p7: an epitope region extending from amino acid region390-41 and 438-443.

These amino acid sequences fail to elicit an immune response in humanswhen contacted through infection or naturally through the environmentbut do elicit an immune response in other mammalian species.

The purified and treated proteins were enriched and further purifiedusing preparative SDS-PAGE electrophoresis and when desired, byimmunoaffinity chromatography employing commercially available (ICNCosta Mesa, Calif. and Advanced Biot.ec.inologies, Columbia, Md.)monoclonal antibodies to gp120, gp41, pp 66/55, p24, p17 and p10 eachindividually coupled to Sepharose CL4B using procedures known in theart. Following purification, each purified HIV protein peptide ofinterest was individually conjugated to the MDP microparticle asdescribed in Example 4. HIV-MDP microparticle conjugates were formulatedas a cocktail containing equal molar concentrations of each HIVprotein/peptide at a final concentration of approximately 1 mgprotein/ml in physiological saline. The HIV-MDP cocktail (0.5 ml) wasemulsified in squalene (1.5 ml containing 0.05% Tween 80) and injectedsubcutaneously into 60 goats at 4 to 6 injection sites per goat. Boosterimmunizations were given at monthly intervals to achieve the desiredantibody responses. Monthly serum samples were obtained from each goatpreceding the monthly booster and tested by Western Blot analysis, HIVneutralization and enzyme immunoassay disclosed in Example 8.6. Once thedesired antibody response was achieved, goats were plasmapheresed fromthe jugular vein employing a Baxter A 201-A401 Autopheresis machine. Thegoat red blood cells were returned in physiological saline. The volumeof plasma collected from each animal at each plasmapheresis was 350ml±5.0 ml.

The Caprine immune plasma was fractionated with octanoic acid, afteradjustment of the pH to 4.8 with hydrochloric acid. The mixture wascentrifuged, filtered and passed through an activated charcoal filter toremove agglomerates and reduce the octanoic acid level to below 0.05%.The immunoglobulin (IgG) containing fraction was further purified bypassage through a series of columns:

1. Sephadex G25 with 20 mM phosphate buffer

2. Ion exchange with Whatman DE53 equilibrated in phosphate buffer

3. Sephadex G25 equilibrated in 0.9% saline

The final column filtrate was sterile filtered and then eitherconcentrated or diluted to give a final concentration of 10 mg IgG/mlwith the desired biological activity. SDS-PAGE and Western Blot analysisdemonstrated reactivity of caprine IgG immunoglobulins at a 1:100dilution with the seven distinct viral proteins as gp120, gp41, p51/66,p24, p17, p7 and p10. Analysis for HIV neutralization demonstrated broadneutralization of HIV1 laboratory and wild strains as disclosed inExample 8.3. Analysis by the enzyme linked immunoassay disclosed inExample 8.3 demonstrated positive reactions to the desired nine epitopesat sample dilutions of 1:1000 or greater. Production lots meeting thesecriteria were given the trade name of HRG214 as disclosed and consistedof sterile, pyrogen free caprine IgG at a concentration of 10 mg/mlsuspended in 0.9% sodium chloride without excipients. Each productionlot is compared to the reference lot as disclosed herein to determineequivalence of HRG214 activity. Potency is expressed as mgequivalent/ml.

Males and non pregnant females over the age of 18 were selected based ontheir having a serodiagnosis of HIV infection documented by WesternBlot, with AIDS defining criteria, clinically symptomatic, and with aCD4 T lymphocyte count<300 cells/μl within 30 days prior to study entry.Clinical and laboratory parameters were used to evaluate efficacy.Clinical parameters included changes in opportunistic infections,changes in body weight, changes in gastrointestinal function, includingstool consistency and frequency, changes in energy level, changes inappetite, physical strength and endurance, and an overall change in thequality of life. Laboratory parameters included changes in CD4 and CD8lymphocyte numbers, selected hematology, blood chemistry and urinalysis,and, when available, changes in viral loads by measuring viral RNA byPCR.

The study results showed that with the use of the inmunochemicallydesigned antibodies patients improved clinically, with decreases inopportunistic infections, increases in body weight, changes ingastrointestinal function, including less severe diarrhea, increases inenergy level, increases in appetite, improvements in physical strengthand endurance, and an overall improvement in the quality of life.Laboratory parameters showed improvements, with increases in CD4 and CD8lymphocyte numbers, improvements in hematology, blood chemistry, andurinalysis numbers, as well as decreases in viral loads by measuringviral RNA by PCR and decreases in infectivity when measured by TCID.

The detailed laboratory results of the study are set forth in theattached Tables 9.1-9.13:

Example 9.1

Clinical Evaluation of the Toxicity and Efficacy of HPG214 in TreatingHIV-Infected Patients

Thirty-five HIV infected patients were treated with HRG214. HRG214 wasevaluated for efficacy in reducing HIV viral burden and amelioratingdisease progression and symptoms. Patients were stratified by CD4number/mm³ (<200 and ≧200) and therapeutic regimen:

Group 1: HRG214, CD4<200, n=11;

Group 2: HRG214 and monthly retreatment, CD4<200, n=6;

Group 3: HRG214 and retreatment at progression, CD4<200, n=5;

Group 4: HRG214, CD4>200, n=7;

Group 5: HPG214 and retreatment at progression (patients receivingchemotherapy for malignancy), CD4<200, n=6;

Group 6: Placebo control, CD4>500, n=19;

Group 7: Placebo control, CD4>200-500, n=3.

Patients in treatment groups received 21-28 daily infusions of HRG214 at1-1.5 mg/kg body weight. Patients with CD4<200 were either retreated ×3days monthly (n=6) or retreated with evidence of HIV progression (n=11).Adverse reactions were limited to minor fever of <2° F., chills,headache, and muscle ache. Clinical chemistry and hematologymeasurements during and after treatment remained unchanged or improvedin all patients over a 90 day period. Twenty-eight patients wereevaluated for change in nutritional status. Twenty-four gained 2-22 lbs.body weight with a mean increase of 4.4 lbs. (p=0.0014). Four of the sixpatients receiving systemic chemotherapy for malignancy remained stable(n=2) or lost weight (2 and 6 lbs respectively). Weight increasescorrelated directly with increases in serum total protein and albuminmeasurements. Quantitative HIV-RNA decreased in all treatment groups.

Group #: Days followed CD4# CD8# HIV RNA Total = n, Survivor = SPre/Post Pre/Post % Change Group 1 ^(HRG214): 153/156 601/699 −18% Day150, n = 11, S = 4 Group 2 ^(HRG214):  44/168  477/1143 −94% Day 345, n= 6,  S = 6 Group 3 ^(HRG214+): 25/67 705/856 −67% Day 469, n = 5,  S =3 Group 4 ^(HRG214): 315/487  970/1164 −94% Day 405, n = 7,  S = 7 Group5 ^(HRG214+): 43/41 476/592 −54% Day 469, n = 6,  S = 2 ^(Chemo) Group 6^(control): 909/628 NA NA Day 469, n = 19 Group 7 ^(control): 315/177 NANA Day 469, n = 3

The rate of CD4/mm³ loss with time in all treatment groups was reduced(p<0.01) compared to control groups 6 and 7. A sustained CD4 increasewas observed in groups 2, 3 and 4. Infectivity measurements bymicroculture (TCID) demonstrated a 2 log reduction in infectivity bytreatment day 7-14 (p<0.001) which was not obvious from quantitativeHIV-RNA measurements. Clinical changes included increases in appetiteand stamina with marked improvements in chronic fatigue syndrome,diarrhea, malabsorption, candidiasis, CMV (retinitis excluded), Herpessimplex and zoster, cutaneous Molluscum contagiosum, oral hairyleukoplakia, wasting syndrome, bacterial folliculitis and pneumonitisand HIV related peripheral neuropathy were observed.

HRG214 offers a new drug to assist in the management of HIV infection.

Data for patient groups 2-5 are presented in more detail below:

Patient Group 2

Patient n=6

Primary Objective: Evaluate safety and efficacy of HRG214 treatment ofHIV infection at a daily dose of 1.5 mg/kg/day for 28 days and monthlyretreatment ×3. Clinical follow-up will continue for 3 years. Patientswill have retreat options with recurrence.

End Points: Normalization of clinical and laboratory parametersincluding improvement in opportunistic infections, incidence ofinfections, wasting syndrome, peripheral neuropathy and improvement inabnormal blood chemistry and hematology, CD4 and CD8 and reductions inHIV-RNA quantitated by PCR.

Safety variables include: Blood chemistry and hematology and clinicalparameters.

Efficacy variable include: HIV-RNA Quantitative by PCP, CD4 and CD8counts.

Follow up period=3 years

Follow-up period to date=>345 days

Study Demographics: Patient n=6; Start Date-Oct. 23, 1995; As of day390, Survivors=6; Deaths=0; Lost to follow up=0.

Patient Demographics: HIV positive, AIDS defining criteria with CD4number<50/mm³.

Treatment Drug(s): HRG-214-1.5 mg/kg/day with monthly retreatments.

TABLE 9.2 Patient Group 2 HIV-RNA QUANTITATIVE BY PCR DAY DAY DAY DAYTEST DAY 1 21-28 60-90 120-150 330-390 Mean 4918.3 2092.5 1338 731.8696.6 Std error 2422.6 1222 927.2 433.1 331.2 Maximum 10649 4880 39931825 1164 Minimum 494 23 19 17 15 Median 4265 1733.5 670 542.5 466

TABLE 9.3 Patient Group 2 QUANTITATIVE CD4/mm³ DAY DAY DAY DAY TEST DAY1 21-28 60-90 120-150 330-390 Mean 44 52.8 52.8 109.3 167.8 Std error14.2 19.1 16.8 33.2 41.7 Maximum 72 96 82 154 189 Minimum 5 4 5 11 23Median 49.5 55.5 62 136 139

TABLE 9.4 Patient Group 2 QUANTITATIVE CD8/mm³ DAY DAY DAY DAY TEST DAY1 21-28 60-90 120-150 330-390 Mean 477.5 437 603.3 911.8 1143 Std error157.1 171.8 173.6 228.9 286.7 Maximum 869 883 804 1376 1752 Minimum 14568 84 319 576 Median 448 398.5 762.5 976 1293

Patient Group 3

Patient n=5

Primary Objective: Evaluate safety and efficacy of HRG214 treatment ofHIV infection at a daily dose of 1.5 mg/kg/day for 28 days. Clinicalfollow-up will continue for 3 years. Patients will have retreat optionswith recurrence.

End Points: Normalization of clinical and laboratory parametersincluding improvement in opportunistic infections, incidence ofinfections, wasting syndrome, peripheral neuropathy and improvement inabnormal blood chemistry and hematology, CD4 and CD8 and reductions inHIV-RNA. quantitated by PCR.

Safety variables include: Blood chemistry and hematology and clinicalparameters.

Efficacy variable include: HIV-RNA Quantitative by PCR, CD4 and CD8counts.

Follow up period=3 year

Follow-up period to date=>469 days

Start date of Jun. 13, 1995; as of day 380, Survivors=3; Deaths=2; Lostto follow up=0

Patient population: HIV positive patients with AIDS defining criteria.Five (5) of the five (5) patients had CD4<50/mm³ blood. HIV-RNAquantitated by PCR demonstrated statistically significant reductionsfollowing treatment; day 7 (P=0.0179) and days 21-28 (P=0.043). HIV RNAmeasurements on days 60-90 and 120-150 demonstrated reduced butincreasing HIV RNA values. All 5 patients were retreated (threeconsecutive doses monthly starting between days 120-150). Followingretreatment a statistically significant (P=0.0006) fall in HIV RNAmeasurements were observed by Day >250. Statistical analysis wasperformed using paired t-Test, Wilcoxon Signed Rank Test andMann-Whitney Rank Sum Test.

START DATE: Jun. 13, 1995

END DATE: Open

TABLE 9.5 Patient Group 2 HIV-RNA QUANTITATIVE BY PCR DAY DAY Day DayDAY TEST DAY 1 DAY 7 21-28 60-90 180-210 240-300 >380 Mean 21911 10643.210962.6 15231.8 18424 9385.2 8217 Std error 5003.7 3244.3 2222.5 7385.95473.5 5436.3 5135 Maximum 41182 21223 17679 41922 35129 30412 36412 Minimum 12292 4448 3790 2984 4017 1036  756 Median 18976 6317 11570 678716854 4708 4926

TABLE 9.6 Patient Group 3 QUANTITATIVE CD4/mm³ DAY DAY Day Day DAY TESTDAY 1 DAY 7 21-28 60-90 180-210 240-300 >380 Mean 25 30.8 13.8 35 40.459.6 67 Std error 6.47 8.16 3.01 9.66 13.39 25.72 33.74 Maximum 42 51 2060 82 154 178 Minimum 9 8 3 10 8 13 16.01 Median 26 24 16 42 28 36 42.12

TABLE 9.7 Patient Group 3 QUANTITATIVE CD8/mm³ DAY DAY Day Day DAY TESTDAY 1 DAY 7 21-28 60-90 180-210 240-300 >380 Mean 705.6 633.8 486.4681.4 570.8 826.6 856.24 Std 137.8 168.2 86 229 120.6 200.9 226 errorMaximum 1152 1037 700 1380 756 1320 1346 Minimum 444 220 260 180 101 169556 Median 585 828 540 720 638 812 843

Patient Group 4

Patient n=7

Primary Objective: Evaluate toxicity and efficacy of HRG214 treatment ofHIV infection at a daily dose of 1.5 mg/kg/day for 28 days with IFNinducer on days 1-7 and 21-23. Clinical follow-up will continue for 3years. Patients will have retreat options with recurrence.

End Points: Normalization of clinical parameters and laboratoryparameters including improvement in OI, incidence of infections, wastingsyndrome, etc., in abnormal blood chemistry and hematology, CD4 and CD8,reductions in HIV-RNA quantitated by PCR.

Follow-up period 3 years

Follow-up period to date=>405 days

Study Demographics: Patient n=7; Start Date-Aug. 25, 1995 As of day 390,Survivors=7; Deaths=0; Lost to follow up=0.

Patient Demographics: HIV positive patients with CD4 number >200 withoutAIDS defining criterion. Treatment Drug(s): 1.5 mg/kg/day for 28 days.Patients will have retreat options with recurrence.

TABLE 9.8 Patient Group 4 HIV-RNA QUANTITATIVE BY PCR DAY DAY DAY DAYTEST DAY 1 21-28 60-90 180-210 330-390 Mean 6078.7 964.3 899.7 658 386.2Std error 949.4 639.6 199.1 544 257 Maximum 7936 2207 1257 1202 983Minimum 4808 80 569 114 54.1 Median 5492 606 873 658 432

TABLE 9.9 Patient Group 4 QUANTITATIVE CD4/mm³ DAY DAY DAY DAY TEST DAY1 21-28 60-90 180-210 330-390 Mean 315 302.7 361.3 409.3 486.5 Std error72.6 70.3 66.8 72.9 71.8 Maximum 429 440 460 540 611 Minimum 180 208 234288 301 Median 336 260 390 400 435

TABLE 9.10 Patient Group 4 QUANTITATIVE CD8/mm³ DAY DAY DAY DAY TEST DAY1 21-28 60-90 180-210 330-390 Mean 970.7 867.7 1016 1094 1164 Std error247.7 158.7 378.8 229.5 235.2 Maximum 1464 1180 1770 1550 1672 Minimum685 663 576 820 921 Median 763 760 702 912 986

Patient Group 5

Patient n=6

Primary Objective: Evaluate toxicity and efficacy of HRG214 treatment.Clinical follow-up will continue for 3 years. Patients will have retreatoptions with recurrence.

End Points: Normalization of clinical parameters and laboratoryparameters including improvement in OI, incidence of infections, wastingsyndrome, etc. in abnormal blood chemistry and hematology, CD4 and CD8,reductions in HIV-RNA quantitated by PCR

Follow-up period 3 years

Follow-up period to date=>469 days

Start date of Jun. 13, 1995 as of day 270, Survivors=2; Deaths=4 (2deaths between 180-210, 1 death between 240-270; one death after 270);Lost to follow up=0

Patient Population: HIV positive patients with AIDS defining criteriaincluding CD4<200/mm³ blood and disseminated Kaposi's sarcoma. Patientswere treated with systemic chemotherapy. Two patients died between days180-210 and two patients died after day 240. HIV-RNA quantitated by PCRdemonstrated reductions at days 60-90, 120-150, 180-210 and 240-270(p=0.018).

Statistical analysis was performed using paired t Test Wilcox, SignedRank Test and Mann-Whitney Rank Sum Test.

TABLE 9.11 Patient Group 5 HIV-RNA QUANTITATIVE BY PCR DAY DAY Day DayDAY TEST DAY 1 21-28 60-90 120-150 180-210 240-270 Mean 12483.3 16973.78373.7 8237.8 8287 4717.5 Std 3159.1 6889.1 3081.8 2152.3 2466.6 3609.5error Maxi- 20233 46586 19514 15249 15570 8327 mum Mini- 442 1242 4901358 4708 1108 mum Median 13682 14510 7027 7392 6435 4717.5

TABLE 9.12 Patient Group 5 QUANTITATIVE CD4/mm³ DAY DAY Day Day DAY TESTDAY 1 21-28 60-90 120-150 180-210 240-270 Mean 43.7 20.5 22.3 17.5 12.636 Std 31.46 12.04 8.45 6.76 4.12 Undefined error Maxi- 200 80 48 48 2436 mum Mini- 4 4 2 2 4 36 mum Median 12 9.5 17 11 8 36

TABLE 9.13 Patient Group 5 QUANTITATIVE CD8/mm³ DAY DAY Day Day DAY TESTDAY 1 21-28 60-90 120-150 180-210 240-270 Mean 476.3 303.5 415.8 371326.4 676 Std 152.9 62.7 159.1 100.7 96.2 Undefined error Maxi- 1170 5401152 710 618 676 mum Mini- 98 86 92 62 101 676 mum Median 352 271.5293.5 358 245 676

Bibliography

1. H. Mitsuya, S. Broder, Nature 325, 773-778 (1987).

2. The Molecular Biology of Tumor Viruses, J. Tooze, et al., Eds.(1973).

3. RNA Tumor Viruses. P. Weiss, Ed. (1982).

4. F. Gonzalez-Scarano, P.E. Shoppe, C.E. Calisher, N. Nathanson,Virology 120, 42-53 (1982).

5. S. Matsuno, S. Inouye, Infection and Immunity 39, 155-158 (1983).

6. J. Mathews, J. Roehrig, The Journal of Immunology 129, 2763-2767(1982).

7. M. Robert-Guroff, M. Brown, R. Gallo, Nature 316, 72-74 (1985).

8. R. Weiss, et al., Nature 316, 69-72 (1985).

9. T. Matthews, et al., Proceedings of the National Academy of Sciences83, 9709-9713 (1986).

10. W. Robey, et al., Proceedings of the National Academy of Sciences83, 7023-7027 (1986).

11. L. Lasky, et al., Science 233, 209-212 (1986).

12. D. Zagury, et al., Nature 326, 249-250 (1987).

13. J. McDougal, et al., Science 231, 382-385 (1986).

14. S. Putney, et al., Science 234, 1392-13905 (1986).

15. S. Norley, R. Kurth, The Retroviridae, J. Levy, Ed. (Plenum Press,1994), vol. 5.

16. J. Carlson, JAMA 260, 674-679 (1988).

17. J. Lange, et al., British Medical Journal 292, 228-230 (1986).

18. J. McDougal, et al., Journal of Clinical Investigation 80, 316-324(1987).

19. A. Amadori, A. De Rossi, G. Faulker-Valle, 1. Chieco-Bianchi,Clinical Immunology and Immunopathology 46, 342-351 (1988).

20. A. Amardori, et al., The Journal of Immunology 89, 2146-2152 (1989).

21. E. Barker, S. W. Barnett, L. Stamataos, J. A. Levy, in The Viruses:The Retroviridae J.A. Levy, Ed. (Plenum Press, New York and London,1995), vol. 4, pp. 1-7.

22. J. A. Levy, in HIV and the Pathogenesis of AIDS A. Press, Ed. (ASMPress, Washington, DC, 1994) pp. 1-5.

23. D. F. Nixon, K. Broliden, G. Ogg, P.-A. Broliden, Immunology 76,515-534 (1992).

24. P. Linsley, J. Ledbetter, E. Kinney-Thomas, S.-L Hu, J Virology 62,3695-3702 (1988).

25. M. Thali, et al., J Virology 66, 5635-5641 (1992).

26. A. Benjouard, J. Gluckmna, H. Rochat, L. Montagnier, E. Bahraoui, J.Virology 66, 2473-83 (1992).

27. T. Chanh, et al., The EMBO Journal 5, 3065-71 (1986).

28. J. Homsy, M. Meyer, J. Levy, J. Virology 64, 1437-40 (1990).

29. M. Tremblay, et al. J Immunology 145, 2896-2901 (1990).

30. R. carry, Science 250, 112,-1129 (1990).

31. M. Mackett, G. Smith, B. Moss, Proc. Natl. Acad. Sci. USA 79,7415-7419 (1982).

32. D. Panicali, E. Paoletti, Proc. Natl. Acad. Sci. USA 79, 4927-4931(1982).

33. S.-L. Hu, S. Kosowski, J. Darymple, Nature 320, 537-540 (1986).

34. S. Chakrabarti, M. Robert-Guroff, F. Wong-Staal, R. Gallo, B. Moss,Nature 320, 535-537 (1986).

35. D. G. Kleid, et al., Science 214, 1125-1129 (1981).

36. C. Cabradilla, et al., Bio/Technology 4, 128-133 (1986).

37. Current Protocols in Immunology (John Wiley & Sons, 1995).

38. Remington's Pharmaceutical Science (Mack Publishing Co, Easton, Pa.,ed. 15th, 1990).

39. B. Karpovsky, J. Titus, D. Stephany, D. Segal, Journal ofExperimental Medicine 160, 1686-1701 (1984).

40. P. Cuatrecasas, Advances in Enzymology 36, 29 (1972).

41. P. Tijssen, Practice and Theory of Enzyme Immunoassay (1985).

42. Stewart, Young, Solid Phase Peptide Synthesis (Pierce Chemical Co,ed. 2nd, 1984).

43. J. Tam, et al., Journal American Chemical Society 105, 6442 (1983).

44. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory, 1982).

45. Z. Grabarek, J. Gergely, Analytical Biochemistry 185 (1990).

46. J. Staros, R. Wright, D. Swingle, Analytical Biochemistry 156,220-222 (186).

47. R. Thrikovich, Analytical Biochemistry 79, 135-143 (1977).

48. F. Gelder, et al. Annals of Surgery, 591-599 (1991).

49. M. Fung, et al., J. Virology, 66, 848-56 (1992)

14 24 amino acids amino acid not relevant not relevant peptide NOinternal Human immunodeficiency virus type 1 SF2 1 Lys Gly Thr Arg ArgAsn Tyr Gln His Leu Trp Arg Trp Gly Thr Leu 1 5 10 15 Leu Leu Gly MetLeu Met Ile Cys 20 23 amino acids amino acid not relevant not relevantpeptide NO internal Human immunodeficiency virus type 1 SF2 2 Ala SerAsp Ala Arg Ala Tyr Asp Thr Glu Val His Asn Val Trp Ala 1 5 10 15 ThrHis Ala Cys Val Pro Thr 20 40 amino acids amino acid not relevant notrelevant peptide NO internal Human immunodeficiency virus type 1 SF2 3Arg Val Val Gln Arg Glu Lys Arg Ala Val Gly Ile Val Gly Ala Met 1 5 1015 Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Val Ser 20 2530 Leu Thr Leu Thr Val Gln Ala Arg 35 40 22 amino acids amino acid notrelevant not relevant peptide NO internal Human immunodeficiency virustype 1 SF2 4 Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg TrpGlu 1 5 10 15 Lys Ile Arg Leu Arg Pro 20 28 amino acids amino acid notrelevant not relevant peptide NO internal Human immunodeficiency virustype 1 SF2 5 Leu Tyr Cys Val His Gln Arg Ile Asp Val Lys Asp Thr Lys GluAla 1 5 10 15 Leu Glu Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys 20 25 16amino acids amino acid not relevant not relevant peptide NO internal 6Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro 1 5 1015 21 amino acids amino acid not relevant not relevant peptide NOinternal 7 Lys Thr Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His Ile AlaLys 1 5 10 15 Asn Cys Arg Ala Pro 20 6 amino acids amino acid notrelevant not relevant peptide NO internal Human immunodeficiency virustype 1 8 Lys Ile Trp Ser Ser Gln 1 5 26 amino acids amino acid notrelevant not relevant peptide NO internal Human immunodeficiency virustype 1 9 Arg Ile Gly Gly Gln Leu Lys Glu Ala Leu Leu Asp Thr Gly Ala Asp1 5 10 15 Asp Thr Val Leu Glu Glu Met Asn Leu Pro 20 25 42 amino acidsamino acid not relevant not relevant peptide NO internal Humanimmunodeficiency virus type 1 10 Gly Leu Lys Lys Lys Lys Ser Val Thr ValLeu Asp Val Gly Asp Ala 1 5 10 15 Tyr Phe Ser Val Pro Leu Asp Lys AspPhe Arg Lys Tyr Thr Ala Phe 20 25 30 Thr Ile Pro Ser Ile Asn Asn Glu ThrPro 35 40 37 amino acids amino acid not relevant not relevant peptide NOinternal Human immunodeficiency virus type 2 NZ 11 Gln Leu Leu Ile AlaIle Val Leu Ala Ser Ala Tyr Leu Ile His Cys 1 5 10 15 Lys Gln Phe ValThr Val Phe Tyr Gly Ile Pro Ala Trp Arg Asn Ala 20 25 30 Ser Ile Pro LeuPhe 35 20 amino acids amino acid not relevant not relevant peptide NOinternal Human immunodeficiency virus type 1 SF2 12 Gly Ile Val Gly AlaMet Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser 1 5 10 15 Thr Met Gly Ala 2010 amino acids amino acid not relevant not relevant peptide NO internalHuman immunodeficiency virus type 1 SF2 13 Phe Leu Gly Phe Leu Gly AlaAla Gly Ser 1 5 10 17 amino acids amino acid not relevant not relevantpeptide NO internal Human immunodeficiency virus type 2 NZ 14 Leu LeuIle Ala Ile Val Leu Ala Ser Ala Tyr Leu Ile His Cys Lys 1 5 10 15 Gln

What is claimed is:
 1. A composition comprising HIV proteins isolated from a lysate of an HIV isolate which has been treated to remove human HLA class I and class II antigens present in said lysate, wherein said proteins have been deglycosylated and wherein said proteins comprise at least one epitope region which does not elicit an immune response in man when encountered by infection or environmental exposure but does elicit an immune response in at least one non-human mammalian species.
 2. A composition in accordance with claim 1, wherein said epitope region encompasses a neutralizing or inactivating region of said HIV protein.
 3. A composition in accordance with claim 1, wherein said epitope region has an amino acid sequence which corresponds to or immunologically mimics a portion of a human protein amino acid sequence.
 4. A composition in accordance with claim 1, which has been enriched for said epitope region(s).
 5. A composition in accordance with claim 4, wherein said epitope region(s) comprises at least about 25% of said protein.
 6. A composition in accordance with claim 5, wherein said epitope region(s) comprises between about 50% and about 95% of said protein.
 7. A composition in accordance with claim 1, which comprises a mixture of lysates from different HIV isolates.
 8. A composition in accordance with claim 1, which comprises a mixture of lysates from HIV1_(MN), HIV1_(BAL), and HIV2_(NZ).
 9. A composition in accordance with claim 1, wherein said epitope region corresponds to or mimics at least one epitope region of proteins of HIV isolate HIV1_(SF2) which does not elicit an immune response in man when encountered by infection or environmental exposure but does elicit an immune response in at least one other mammalian species.
 10. A composition in accordance with claim 9, wherein said epitope region corresponds to or mimics an epitope region of at least one of the following HIV1_(SF2) proteins: (a) envelope gp120 external glycoprotein; (b) envelope gp41 transmembrane glycoprotein; (c) reverse transcriptase; (d) protease p10; or (e) gag precursor.
 11. A composition in accordance with claim 10, wherein at least one of said epitope regions of HIV_(SF2) proteins comprises: (a) a region identified by SEQ ID NO: 1; (b) a region identified by SEQ ID NO: 2; (c) a region identified by SEQ ID NO: 3; (d) a region identified by SEQ ID NO: 10; (e) a region identified by SEQ ID NO: 9; (f) a region identified by SEQ ID NO: 6; (g) a region identified by SEQ ID NOS: 7 and 8; (h) a region identified by SEQ ID NO: 4; or (i) a region identified by SEQ ID NO:
 5. 12. A composition in accordance with claim 1, which further comprises an adjuvant.
 13. A composition in accordance with claim 12, wherein said adjuvant comprises a carrier molecule to which the HIV protein is coupled.
 14. A composition in accordance with claim 13, wherein said carrier molecule comprises poly-L-lysine, keyhole limpet hemocyanin, thyroglobulin, an albumin or tetanus toxoid.
 15. A composition in accordance with claim 13, wherein said carrier molecule comprises multiple repeats of a glycopeptide.
 16. A composition in accordance with claim 15, wherein said carrier molecule comprises multiple repeats of muramyl dipeptide.
 17. A composition in accordance with claim 16, wherein said multiple repeats of muramyl dipeptide are crosslinked.
 18. A composition in accordance with claim 17, wherein said multiple repeats of muramyl dipeptide comprise a terminal dipeptide of L-alanine-D-isoglutamine.
 19. A composition comprising a synthetic peptide which comprises an epitope region which corresponds to or mimics a neutralizing or inactivating region of an HIV protein, wherein said peptide does not elicit an immune response in humans when encountered by infection or environmental exposure but does elicit an immune response in at least one non-human mammalian species.
 20. A composition in accordance with claim 19, wherein said epitope region has an amino acid sequence which corresponds to or mimics a portion of a human protein.
 21. A composition in accordance with claim 19, wherein at least one amino acid within said epitope region is modified to enhance MHC interactions or the immune response obtained following administration of said peptide to a non-human mammal.
 22. A composition in accordance with claim 21, wherein at least one amino acid is modified so as to create an amphipathic helix with said epitope region bracketed between hydrophilic amino acids and hydrophobic amino acids.
 23. A composition in accordance with claim 19, comprising a mixture of said synthetic peptides, wherein said peptides comprise epitope regions which correspond to or mimic more than one neutralizing or inactivating region of HIV proteins.
 24. A composition in accordance with claim 19, wherein said epitope region corresponds to or mimics a neutralizing or inactivating region of a protein of HIV isolate HIV1_(SF2).
 25. A composition in accordance with claim 23, wherein said epitope regions correspond to or mimic more than one neutralizing or inactivating region of proteins of HIV isolate HIV1_(SF2).
 26. A composition in accordance with claim 24 or 25, wherein said HIV1_(SF2) protein comprises: (a) envelope gp120 external glycoprotein; (b) envelope gp41 transmembrane glycoprotein; (c) reverse transcriptase; (d) protease p10; or (e) gag precursor.
 27. A composition in accordance with claim 26, wherein said neutralizing or inactivating region of HIV_(SF2) protein comprises: (a) a region identified by SEQ ID NO: 1; (b) a region identified by SEQ ID NO: 2; (c) a region identified by SEQ ID NO: 3; (d) a region identified by SEQ ID NO: 10; (e) a region identified by SEQ ID NO: 9; (i) a region identified by SEQ ID NO: 6; (g) a region identified by SEQ ID NOS: 7 and 8; (h) a region identified by SEQ ID NO: 4; or (i) a region identified by SEQ ID NO:
 5. 28. A composition in accordance with claim 19, which further comprises an adjuvant.
 29. A composition in accordance with claim 28, wherein said adjuvant comprises a carrier molecule to which the HIV peptide is coupled.
 30. A composition in accordance with claim 29, wherein said carrier molecule comprises poly-L-lysine, keyhole limpet hemocyanin, thyroglobulin, an albumin or tetanus toxoid.
 31. A composition in accordance with claim 29, wherein said carrier molecule comprises multiple repeats of a glycopeptide.
 32. A composition in accordance with claim 31, wherein said carrier molecule comprises multiple repeats of muramyl dipeptide.
 33. A composition in accordance with claim 32, wherein said multiple repeats of muramyl dipeptide are crosslinked.
 34. A composition in accordance with claim 33, wherein said multiple repeats of muramyl dipeptide comprise a terminal dipeptide of L-alanine-D-isoglutamine.
 35. A method of identifying a neutralizing or inactivating region of an HIV protein, wherein said neutralizing or inactivating region does not elicit an immune response in man when encountered by infection or environmental exposure but does elicit an immune response in a non-human mammal, which comprises: (a) extracting HIV proteins from a lysate of an HIV strain; (b) immunizing a non-human mammal with said extract; (c) obtaining antisera from said immunized mammal; (d) employing said antisera in a competitive immunoassay with human HIV antisera to identify regions of HIV proteins which are recognized by antibodies in said antisera but not recognized by antibodies in said human antisera; and (e) determining which of said regions is a neutralizing or inactivating region.
 36. A composition in accordance with claim 35, wherein said neutralizing or inactivating region comprises or is homologous to one of the following regions of a protein of HIV isolate HIV1_(SF2): (a) a region identified by SEQ ID NO: 1; (b) a region identified by SEQ ID NO: 2; (c) a region identified by SEQ ID NO: 3; (d) a region identified by SEQ ID NO: 10; (e) a region identified by SEQ ID NO: 9; (f) a region identified by SEQ ID NO: 6; (g) a region identified by SEQ ID NOS: 7 and 8; (h) a region identified by SEQ ID NO: 4; or (i) a region identified by SEQ ID NO:
 5. 37. A composition comprising the proteins of claim 1 in combination with a pharmaceutically acceptable carrier.
 38. A composition in accordance with claim 37, wherein said proteins are coupled to a macromolecular carrier.
 39. A composition in accordance with claim 38, wherein said carrier is a microparticle of muramyl dipeptide.
 40. A composition comprising one or more synthetic peptides of claim 19 in combination with a pharmaceutically acceptable carrier.
 41. A composition in accordance with claim 40, wherein said peptides are coupled to a macromolecular carrier.
 42. A composition in accordance with claim 41, wherein said carrier is a microparticle of muramyl dipeptide.
 43. A composition comprising viral proteins isolated from a viral lysate which has been treated to remove human HLA class I and class II antigens present in said lysate, wherein said proteins have been deglycosylated and wherein said proteins comprise at least one epitope region which does not elicit an immune response in man when encountered through infection environmental exposure but does elicit an immune response in at least one non-human mammalian species.
 44. A composition in accordance with claim 43, wherein said epitope region encompasses a neutralizing or inactivating region of said viral protein.
 45. A composition in accordance with claim 43, wherein said epitope region has an amino acid sequence which corresponds to or immunologically mimics a portion of a human protein amino acid sequence.
 46. A composition in accordance with claim 43, wherein said proteins are coupled to a macromolecular carrier.
 47. A composition in accordance with claim 46, wherein said carrier is a muramyl dipeptide microparticle.
 48. A composition in accordance with claim 46, wherein said muramyl dipeptide comprises a terminal dipeptide of L-alanine-D-isoglutamine.
 49. A composition comprising a synthetic peptide which comprises an epitope region which corresponds to or mimics a neutralizing or inactivating region of a viral protein, wherein said peptide does not elicit an immune response in humans when encountered through infection or environmental exposure but does elicit an immune response in at least one non-human mammal.
 50. A composition in accordance with claim 49, wherein said epitope region has an amino acid sequence which corresponds to or immunologically mimics a portion of a human protein amino acid sequence.
 51. A composition in accordance with claim 49, wherein said proteins are coupled to a macromolecular carrier.
 52. A composition in accordance with claim 51, wherein said carrier is a muramyl dipeptide microparticle.
 53. A composition in accordance with claim 51, wherein said muramyl dipeptide comprises a terminal dipeptide of L-alanine-D-isoglutamine.
 54. A method for identifying a neutralizing or inactivating region of a viral protein, wherein said neutralizing or inactivating region does not elicit an immune response in man when encountered through infection or environmental exposure but does elicit an immune response in a non-human animal, which comprises: (a) extracting viral proteins from a viral lysate; (b) immunizing a non-human mammal with said extract; (c) obtaining antisera from said immunized mammal; (d) employing said antisera in a competitive immunoassay with human viral antisera to identify regions of viral proteins which are recognized by antibodies in said antisera but not recognized by antibodies in said human sera; and (e) determining which of said regions is a neutralizing or inactivating region. 